Preparation of Ligand-Free TiO2 (Anatase) Nanoparticles through a

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Langmuir 2008, 24, 6988-6997

Preparation of Ligand-Free TiO2 (Anatase) Nanoparticles through a Nonaqueous Process and Their Surface Functionalization T. Kotsokechagia,† F. Cellesi,*,† A. Thomas,‡ M. Niederberger,§ and N. Tirelli*,† Laboratory for Polymers and Biomaterials, School of Pharmacy and Pharmaceutical Sciences, UniVersity of Manchester, Manchester M13 9PT, United Kingdom, School of Physics and Astronomy, Photon Science Institute, UniVersity of Manchester, Manchester M13 9PL, United Kingdom, and Laboratory for Multifunctional Materials, Department of Materials, ETH Zu¨rich, Wolfgang-Pauli-Strasse 10, 8093 Zu¨rich, Switzerland ReceiVed February 13, 2008. ReVised Manuscript ReceiVed March 28, 2008 We here present a new method for preparing ligand-free titania nanoparticles, which are easily amenable to surface functionalization in an aqueous environment. The specific advantage of this method is that it combines the advantages of nonaqueous synthetic processes (high crystallinity) to those of a surface functionalization in a water medium, which allows for a wider variety of biofunctional (and nonorganic-soluble) groups to be added on the nanoparticles. In particular, we report on the characterization of the three phases of synthesis, dispersion in water environment and surface functionalization of the nanoparticles, focusing on a qualitative evaluation of the surface adsorption mechanism.

Introduction In the vast range of applications of titanium dioxide, e.g., (photo)catalysis, optics, photoconductivity, gas sensing, paints, and cosmetics1–3 but also prosthetics and implants,4 its functional (optical, chemical) properties are mostly determined by the exposed surface area of the material, which, being related to surface/volume ratio, has a peak at the nanoscale.5 Indeed crystalline TiO2 nanoparticles have been successfully used for antimicrobial coatings of medical devices,6 due to their UV-activated self-cleaning and disinfectant properties; it is noteworthy that the anatase crystalline form is particularly interesting, since it has both the highest reactivity in photocatalysis7 and the best performance in terms of antimicrobial activity.8 We are interested in crystalline (anatase) TiO2 nanoparticles since they offer the possibility to combine medical treatment (oxidizing properties) and imaging (radio-opacity, that allows easy imaging of TiO2 artifacts, e.g., through computed tomography9), but appropriate preparative techniques must be available for (a) producing them in a size range 5-200 nm (higher than renal excretion limit and low enough to surely prevent embo* Corresponding authors. E-mail: [email protected]; nicola. [email protected]. † Laboratory for Polymers and Biomaterials, School of Pharmacy and Pharmaceutical Sciences, University of Manchester. ‡ School of Physics and Astronomy, Photon Science Institute, University of Manchester. § Laboratory for Multifunctional Materials, Department of Materials, ETH Zu¨rich. (1) Diebold, U. Surf. Sci. Rep. 2003, 48(5-8), 53–229. (2) Hagfeldt, A.; Gratzel, M. Chem. ReV. 1995, 95(1), 49–68. (3) Braun, J. H.; Baidins, A.; Marganski, R. E. Prog. Org. Coat. 1992, 20(2), 105–138. (4) Li, J. G. Biomaterials 1993, 14(3), 229–232. (5) Niederberger, M.; Garnweitner, G.; Buha, J.; Polleux, J.; Ba, J. H.; Pinna, N. J. Sol-Gel Sci. Technol. 2006, 40(2-3), 259–266. (6) Song, J. S.; Lee, S.; Cha, G. C.; Jung, S. H.; Choi, S. Y.; Kim, K. H.; Mun, M. S. J. Appl. Polym. Sci. 2005, 96(4), 1095–1101. (7) Tanaka, K.; Capule, M. F. V.; Hisanaga, T. Chem. Phys. Lett. 1991, 187(1-2), 73–76. (8) Machida, M.; Norimoto, K.; Kimura, T. J. Am. Ceram. Soc. 2005, 88(1), 95–100. (9) Utomo, M. B.; Warsito, W.; Sakai, T.; Uchida, S. Chem. Eng. Sci. 2001, 56(21-22), 6073–6079.

lization in capillary networks10) and with narrow dispersity, in order to ensure a fine control of the functional properties, and (b) functionalizing their surface for providing targeting and avoiding agglomeration: since TiO2 nanoparticles have an isoelectric point at pH ) 5-7 in water,11 their low ζ potential under physiological conditions does not offer sufficient electrostatic stabilization to prevent particle flocculation or precipitation. A large number of preparative methods exist for metal oxide nanoparticles; powder-based techniques and aqueous sol-gel processes (such as metal alkoxide hydrolysis) are among the most popular, but they generally fail to provide good control over at least one of the above-mentioned criteria, i.e., crystallinity, size, and surface composition (a good overview of these problems is provided by Niederberger and Garnweitner12). Nonaqueous methods (mostly based on reactions of metal halides or alkoxides with organic molecules in solution, or between themselves12,13) have been demonstrated to overcome a number of these problems. For example the decomposition of titanium tetrachloride in benzyl alcohol can provide (via benzyl chloride elimination) well-defined anatase nanoparticles,14,15 whose surface is covered by benzyloxy groups; if stronger ligands, e.g., enediols such as dopamine or 4-tert-butyl catechol, are added during the process, they displace benzyloxy groups and provide the nanoparticles with a ligandcovered surface.16 It is noteworthy that this preparative route not only offers the possibility to control crystal growth without the use of surfactants but is also based on a widely accepted and well-tolerated solvent: benzyl alcohol has a low toxicity and is already approved for use in food and fragrances.17 (10) Gupta, A. K.; Gupta, M. Biomaterials 2005, 26(18), 3995–4021. (11) Gumy, D.; Morais, C.; Bowen, P.; Pulgarin, C.; Giraldo, S.; Hajdu, R.; Kiwi, J. Appl. Catal., B 2006, 63(1-2), 76–84. (12) Niederberger, M.; Garnweitner, G. Chem.sEur. J. 2006, 12(28), 7282– 7302. (13) Padmanabhan, S. C.; Pillai, S. C.; Colreavy, J.; Balakrishnan, S.; McCormack, D. E.; Perova, T. S.; Gun’ko, Y.; Hinder, S. J.; Kelly, J. M. Chem. Mater. 2007, 19(18), 4474–4481. (14) Niederberger, M.; Bartl, M. H.; Stucky, G. D. J. Am. Chem. Soc. 2002, 124(46), 13642–13643. (15) Niederberger, M.; Bartl, M. H.; Stucky, G. D. Chem. Mater. 2002, 14(10), 4364–4370. (16) Niederberger, M.; Garnweitner, G.; Krumeich, F.; Nesper, R.; Colfen, H.; Antonietti, M. Chem. Mater. 2004, 16(7), 1202–1208. (17) Bindu, N. Int. J. Toxicol. 2001, 20, 23–50.

10.1021/la800470e CCC: $40.75  2008 American Chemical Society Published on Web 06/04/2008

Preparation of Ligand-Free TiO2

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Scheme 1. The Synthesis of TiO2 Anatase Nanoparticles Accomplished by the in Situ Generation and Condensation of Ti Tetraethoxide in Benzyl Alcohol, Whose Condensations Generates Nanoparticles Mostly Covered with Ethoxy Groupsa

a

These nanoparticles could then be isolated and later functionalized with appropriate hydrophilic ligands.

have also allowed the functionalization of titania nanoparticles24 and allowed to tune the assembly of complex organic-inorganic architectures.25

We have here modified this method into a two-step process, which is articulated in a first nonaqueous phase (advantages: good control over crystallinity and size) followed by a waterbased procedure where hydrolyzable surface groups are removed to yield “ligand-free” or “naked” nanoparticles. Among the various advantages arising from this preparative method there is the possibility to easily prepare “bar-coded” nanoparticles, where a parent system provides identical size or bulk properties to a library of nanoparticles differing in surface composition, e.g., displaying different bioactive and fluorescent groups. The core of this method is the presence of hydrolyzable groups on the titania surface after the nonaqueous process; more specifically, residues that are easily hydrolyzable at acidic or basic pH, i.e., where titania has a positive or negative ζ potential and therefore the resulting nanoparticles are electrostatically stabilized against agglomeration. Benzyl oxides, however, are not very suitable groups, since their hydrophobic nature should cause rapid particle aggregation rather than hydrolysis. We have therefore modified the previously presented benzyl alcohol-based protocol,14,15 through a prealkoxylation of TiCl4 in ethanol, followed by its nonhydrolytic decomposition and TiO2 formation in benzyl alcohol (Scheme 1). This process may be assisted by benzyl alcohol, but we hypothesize that the lower steric hindrance and therefore the likely higher ligand strength of ethoxy groups should provide a mostly ethoxy-coated surface, with little presence of benzyl residues. Once in water and at an appropriately low pH, these groups would be rapidly hydrolyzed, providing positively charged, substantially “naked” nanoparticles. This mostly ligand-free surface would then allow the adsorption of ligands; we here have polarized our attention onto strong, chelating ones (enediols) that are generally assumed to react with gem-titanols.18–20 This process could allow a precise biofunctionalization of titana nanoparticles, similarly to what already accomplished to macroscopic surfaces.21–23 Such ligands bound to polymer backbones

Materials. Benzyl alcohol (anhydrous, 99.8%), titanium(IV) chloride (TiCl4, purity g99.0%), dopamine hydrochloride, and standard 0.1 N hydrochloric acid solution were supplied by SigmaAldrich (Buchs, Switzerland). Ethanol (purity >99.7%) and diethyl ether were purchased from BDH (U.K.). Water was predistilled and further purified by a Milli-Q system (Millipore, U.K.). Preparation of Nanoparticles. One milliliter of TiCl4 (9.12 × 10-3 mol) was slowly introduced to a glass vial containing 5 mL of anhydrous ethanol, producing a completely transparent yellow solution. Twenty milliliters of anhydrous benzyl alcohol (0.19 mol) was added, and the container was left open with the whole mixture at 80 °C and continuously stirred for about 9-10 h until the average particle size of the nanoparticles had reached 8-9 nm (dynamic light scattering analysis in benzyl alcohol; correspondingly the suspension turns slightly opaque). The suspension was precipitated in 80 mL of diethyl ether, centrifuged (3500 rpm for 5 min), and separated by decantation. The white precipitate was then washed in diethyl ether and centrifuged again. The precipitate was then redispersed in 20 mL of a water-ethanol mixture (50:50 w/w) brought to pH ) 1 using a 0.1 M HCl solution. The resulting suspension was dialysed in purified water at pH 1 (by HCl) using a dialysis membrane with MWCO 100000 g/mol. The final concentration of the suspension (typically 1% w/w) was calculated by freeze-drying 1 mL of sample and weighing the solid at the end of the process. Characterization. Attenuated Total Reflection Infrared Spectroscopy (ATR-IR). FT-IR spectra were recorded in ATR mode on a Tensor 27 Brucker spectrometer. Dynamic Light Scattering (DLS) Analysis and ζ-Potential Measurements. These were performed by a Zetasizer Nano ZS Instrument (Malvern Instrument Ltd., U.K.), with a back-scattered light detection (173° detection optics) and a 633 nm HeNe laser beam, and connected to a Malvern autotritator MPT-2. Raw data were processed by Malvern DTS software.

(18) Dimitrijevic, N. M.; Saponjic, Z. V.; Bartels, D. M.; Thurnauer, M. C.; Tiede, D. M.; Rajh, T. J. Phys. Chem. B 2003, 107(30), 7368–7375. (19) Rajh, T.; Chen, L. X.; Lukas, K.; Liu, T.; Thurnauer, M. C.; Tiede, D. M. J. Phys. Chem. B 2002, 106(41), 10543–10552. (20) Moser, J.; Punchihewa, S.; Infelta, P. P.; Gratzel, M. Langmuir 1991, 7(12), 3012–3018. (21) Dalsin, J. L.; Lin, L. J.; Tosatti, S.; Voros, J.; Textor, M.; Messersmith, P. B. Langmuir 2005, 21(2), 640–646.

(22) Dalsin, J. L.; Hu, B. H.; Lee, B. P.; Messersmith, P. B. J. Am. Chem. Soc. 2003, 125(14), 4253–4258. (23) Fan, X. W.; Lin, L. J.; Messersmith, P. B. Biomacromolecules 2006, 7(8), 2443–2448. (24) Tahir, M. N.; Eberhardt, M.; Theato, P.; Faiss, S.; Janshoff, A.; Gorelik, T.; Kolb, U.; Tremel, W. Angew. Chem., Int. Ed. 2006, 45(6), 908–912. (25) Meuer, S.; Oberle, P.; Theato, P.; Tremel, W.; Zentel, R. AdV. Mater. 2007, 19(16), 2073.

Experimental Section

6990 Langmuir, Vol. 24, No. 13, 2008 ThermograVimetric Analysis (TGA). About 10 mg of freeze-dried TiO2 nanoparticle samples was analyzed on a Q5000IR TGA thermobalance (TA Instruments, Delaware USA). Thermogravimetric curves were collected at 10 °C/min scan rate with an isotherm at 120 °C (for 60 min or more) in order to quantify the amount of physisorbed water. X-ray Photoelectron Spectroscopy (XPS). The ESCA300 instrument at Daresbury Laboratory was used for XPS analysis in this work. A monochromated Al KR (hν ) 1486.6 eV) source was used in conjunction with a Scienta 300 mm mean radius analyzer. All spectra are charge referenced to adventitious carbon at a binding energy of 285 eV and were recorded at a take off angle of 90° relative to the surface at a pass energy of 150 eV. The base pressure of the ultrahigh vacuum (UHV) chamber was 1 × 10-9 mbar. X-ray gun conditions: 14 kV with 0.2 A emission, 2.8 kW; means of instrument calibration (ref materials), Ag foil; area of scan dimensions, 0.5 × 6 mm. X-ray Powder Diffraction (XRD). XRD patterns were measured in reflection mode with Cu KR radiation on a IPD PW1800 diffractometer (PANalytical). Transmission Electron Microscopy (TEM). The measurements for the as-synthesized nanoparticles were performed on a Philipps CM 200 operated at 160 kV equipped with a CCD Camera Gatan. For the characterization of the as-synthesized anatase nanoparticles the wet precipitate after the addition of diethyl ether was redispersed in ethanol and one drop of the transparent dispersion was deposited onto a copper grid covered by an amorphous carbon film. To prevent agglomeration of the nanoparticles, the copper grid was placed on filter paper at the bottom of a Petri dish. The nanoparticles redispersed in water were pipetted on carbon/ Formvar coated 100 mesh grids, which were then washed with distilled water three times, stained in 1% uranyl acetate solution, and finally treated with 1 drop of 1.8% methylcellulose solution. The liquid in excess was removed on filter paper, and the grids were allowed to dry prior to analysis. Specimens were observed in a Technai 12 electron microscope at 100 kV. Atomic Force Microscopy (AFM). AFM analysis was performed using an MFP-3D (Asylum Research, Santa Barbara, California). A water dispersion of titania nanoparticles was deposited on mica, dried at room temperature under atmospheric pressure, and analyzed in tapping mode with an Olympus AC240TS cantilever (small spring constant type silicon probe). Surface Adsorption on Titania Nanoparticles. Dopamine adsorption onto TiO2 nanoparticles was monitored by spectrophotometric analysis using a Perkin-Elmer Lambda 25 UV/vis spectrometer. UV-Vis Experiments. In a typical experiment 1 mL of a 0.74 mg/mL TiO2 nanoparticle-water suspension was diluted in 1 mL of water at pH 1.5 (HCl). The UV-vis absorption spectra (λ ) 250-800 nm) were recorded after each addition of aliquots of a 11.7 mM dopamine solution at 20 °C; before the spectra were collected, the stability of the absorption at 435 nm was checked as an indication that equilibrium was reached. All the spectra were then corrected for removing the scattering component applying an exponential baseline. HPLC Analysis. 0.5 mL of a 0.37 mg/mL TiO2 nanoparticle-water suspension at pH 1.5 (HCl) were mixed with different aliquots of a 11.7 mM dopamine solution. After adsorption equilibrium was reached at 20 °C, dopamine-grafted nanoparticles were precipitated by increasing the pH upon addition of 0.5 mL of 100 mM carbonate/ bicarbonate buffer at pH 8. The precipitated suspension was centrifuged at 8000 rpm for 30 min and the supernatant was collected for analysis. The concentration of unbound dopamine present in the supernatant was measured by a high-performance liquid chromatography (HPLC) system (Laserchrom, U.K.), consisting of a C18 column, a 1% acetic acid-water as mobile phase, and an S3210 UV-vis detector set at 280 nm. HPLC was previously calibrated with dopamine (concentrations ranging from 0.1 to1 mM). Langmuir Model and Surface Adsorption Data Treatment. We have here used the typical assumptions of a Langmuir model: (1) all surface sites have the same adsorption energy for the adsorbate,

Kotsokechagia et al. the same applies to the solvent too; (2) adsorption (of either solvent or adsorbate) at one site does not affect the availability or the energy of adsorption of sites next to it (as they adsorb either solvent or adsorbate); (3) the activity of the adsorbate is directly proportional to its concentration (and a similar statement for the solvent). Accordingly, the Langmuir isotherm equation (extended to solid-liquid systems26,27) is

Γ ) Γmax

KL[Li]eq 1 + KL[Li]eq

(1)

where Γand Γmax are the equilibrium adsorption density and the maximum adsorption density for a ligand Li, respectively, and are expressed in mol/m2, KL is the Langmuir constant (M-1), and [Li]eq is the concentration of the ligand in solution (M) at equilibrium with the adsorbed ligand. Due to the fact that the nanoparticles surface areas are not precisely known, it is convenient to replace the surface densities with volumetric concentrations. Depending on the mechanism (here we will take into account a monomolecular and a bimolecular one), it is possible to replace Γ with the equilibrium concentration of dopamine-titanium complexes, [TiDA]eq or [Ti2DA]eq ()moles of adsorbed ligand/ volume of suspension), and Γmax with the maximum concentration of sites available on the nanoparticles surfaces, [Ti]0 or [Ti]0/2; therefore, using simple algebraic passages, it is possible to obtain the following convenient formulation

1 [TiDA]eq

or

1 1 x x ) + (2) [Ti2DA]eq [Ti]0 [Ti]0 KL[DA]eq

where x ) 1 or 2, respectively, in the monomolecular or bimolecular mechanism. The relation between Langmuir constant and surface adsorption equilibrium constant is easy to demonstrate in both cases, yielding

{

KL )

[TiDA]eq K mono ads ) + [Ti]eq[DA]eq [H ]

(3)

[Ti2DA]eq ) K bi KL ) ads2[Ti]eq [Ti]eq ⁄ 2[DA]eq

Please note that the equilibrium concentrations of the various species are interrelated through the overall mass conservation:

{

[DA]0 ) [DA]eq + [TiDA]eq, [Ti]0 ) [Ti]eq + [TiDA]eq (4) [DA]0 ) [DA]eq + [Ti2DA]eq, [Ti]0 ) [Ti]eq + ⁄2[Ti2DA]eq 1

HPLC Measurements. Measuring the free dopamine concentration in solution, [DA]eq, and knowing its initial concentration, it is possible to express [TiDA]eq or [Ti2DA]eq as their difference

1 1 x x ) + [DA]0 - [DA]eq [Ti]0 [Ti]0 KL[DA]eq

(5)

UV-Vis Measurements. After eliminating the contribution arising from nanoparticle turbidity and correcting the data for the dilution during titration, it is possible to use the Lambert-Beer law to link the surface complex concentration to the UV-vis absorbance: (26) Giammar, D. E.; Maus, C. J.; Xie, L. Y. EnViron. Eng. Sci. 2007, 24(1), 85–95. (27) Oscik, J., Adsorption; Ellis Horwood Ltd.: Chichester, 1982.

Preparation of Ligand-Free TiO2

A ) ε[TiDA]eq or ε[Ti2DA]eq f 1 x 1 x + ) A ε[Ti]0 ε[Ti]0 KL[DA]eq

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(6)

1 x x ) + ε[Ti]0 ε[Ti]0 KL([DA]0 - A ⁄ ε) NOTE: Langmuir models have been already extensively utilized to fit the adsorption behavior of catechols on titania28 and we have therefore successfully applied this model. However, Freundlich isotherms have been recently suggested to provide more accurate, although not significantly different, results.29

Results and Discussion Nonaqueous Preparative Method of “Naked” TiO2 Nanoparticles. Nonhydrolytic sol-gel routes involving thermal condensation reactions, such as that of metal halides with metal alkoxides, are well-known in literature.30,31 For titania preparation, nonhydrolytic pathways generally involve the reaction of titanium tetrachloride with either a metal alkoxide or an organic oxygen donor, such as diisopropyl ether:32 Ti-O-Ti bonds are formed as a result of the condensation of Ti-Cl and the M-OR groups, where M is either a titanium atom31 or an organic alkyl group.33,34 Halide-free reactions (e.g., between Ti alkoxides) have also been reported, for example based on an aprotic condensation mechanism leading to an oxo bridge through the elimination of an organic ether.33 However, in here we utilized a modified nonhydrolytic procedure, which enabled the low-temperature formation of highly crystalline (anatase) nanoparticles with high surface areas14,15 based on the in situ generation of an alkoxide through the reaction between TiCl4 and benzyl alcohol. The resulting nanoparticle surface is covered by benzyloxy groups and/or by stronger ligands (e.g., dopamine or other multidentate structures) that can be introduced in the reaction environment.16 However, despite the well-defined structure and the control over surface composition, such nanoparticles generally cannot be redispersed in water in an individual form (due to hydrophobically induced aggregation and then flocculation), giving rise to more or less ordered aggregates in dependence of the chemistry of the surface groups.35 In our synthetic procedure, we have used ethanol as the oxygen donor group instead of benzyl alcohol, forming in situ ethoxide functions by reaction with titanium chloride. The motivation of this choice is in the hydrolytic lability of Ti ethoxides, which would allow to obtain an organic-free surface by exposure of the nanoparticles to water. Nanoparticle Preparation. Titanium tetrachloride was added very slowly and under vigorous stirring to ethanol, in order to keep under control the potentially violent formation of HCl(g). At the end of gas evolution, the solution is likely to contain titanium ethoxide species formed in situ, possibly Ti(OEt)2Cl2 or oligomers with bridging ethoxy groups and mostly pentaco(28) Rodriguez, R.; Blesa, M. A.; Regazzoni, A. E. J. Colloid Interface Sci. 1996, 177(1), 122–131. (29) Lana-Villarreal, T.; Rodes, A.; Perez, J. M.; Gomez, R. J. Am. Chem. Soc. 2005, 127(36), 12601–12611. (30) Corriu, R. J. P.; Leclercq, D.; Lefevre, P.; Mutin, P. H.; Vioux, A. J. Non-Cryst. Solids 1992, 146(2-3), 301–303. (31) Corriu, R. J. P.; Leclercq, D.; Lefevre, P.; Mutin, P. H.; Vioux, A. J. Mater. Chem. 1992, 2(6), 673–674. (32) Arnal, P.; Corriu, R. J. P.; Leclercq, D.; Mutin, P. H.; Vioux, A. Chem. Mater. 1997, 9(3), 694–698. (33) Niederberger, M.; Garnweitner, G.; Pinna, N.; Neri, G. Prog. Solid State Chem. 2005, 33(2-4), 59–70. (34) Vioux, A. Chem. Mater. 1997, 9(11), 2292–2299. (35) Polleux, J.; Pinna, N.; Antonietti, M.; Hess, C.; Wild, U.; Schlogl, R.; Niederberger, M. Chem.sEur. J. 2005, 11(12), 3541–3551.

Figure 1. Size distribution and average size vs time (curves and data points corresponding to 360, 450, 480, 510, 540, 570 min) during the TiO2 synthesis in benzyl alcohol at 80 °C. The presence of scattering centers becomes evident after 6 h; particle growth then possibly prevails on the nucleation of new particles.

ordinated titanium atoms. The mixture was diluted in benzyl alcohol which was used as reaction medium at a temperature of 80 °C. The ethanol-TiCl4 reaction is very rapid and supposedly complete at the time of the addition of benzyl alcohol (2 min after that of titanium chloride). We hypothesize benzyl alcohol not to substantially replace ethanol as a ligand, because of higher steric hindrance of benzyl residues; however, the exchange of alkoxy ligands around titanium centers is a complex matter: for example, the more sterically hindered isopropoxide residues are only partially replaced by the more nucleophilic ethanol in excess of the latter,36 while the exchange is stoichiometric in the presence of more bulky alcohols.37 We therefore suppose any further reaction to involve titanium tetraethoxide. In summary, partial exchange cannot be excluded. Dynamic light scattering analysis clearly showed the presence of colloidal objects only after 6 h, when they are recorded with a size distribution in the range 4-10 nm (6.5 nm average); we consider the previous period as a “lag/nucleation/early growth time”, during which the small dimensions and/or the low refractive index difference with the solvent (due, e.g., to imperfect crystallization) result in a negligible scattering intensity. In a second phase the particles slowly grow in size (Figure 1) until reaching a critical dimension (above 20 nm) that induces precipitation; prior to that, the overall scattering intensity does not appreciably change throughout this phase and this may indicate a negligible or no increase in the number of scattering particles and that therefore particle growth overwhelms nucleation. This hypothesis could be justified by the nucleation rate dependence on the (decreasing) alkoxide concentration, which should be steeper than that of the growth rate, and seems to be confirmed by dynamic light scattering, which records a shift of the size distribution to larger diameters (apparently nanoparticles smaller than 5 nm are virtually absent after 9 h), but no increase in polydispersity. Since therefore the reaction time seems to be an efficient way to control the particle size, for further experiments we have chosen to fix it at 9 h for producing particles with an average size of (36) Finnie, K. S.; Luca, V.; Moran, P. D.; Bartlett, J. R.; Woolfrey, J. L. J. Mater. Chem. 2000, 10(2), 409–418. (37) Perez, Y.; del Hierro, I.; Fajardo, M.; Otero, A. J. Organomet. Chem. 2003, 679(2), 220–228.

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Figure 3. XRD powder pattern of as-synthesized nanocrystalline anatase particles prepared in benzyl alcohol. The reflections are indexed according to the anatase phase. Figure 2. TEM images of TiO2 nanoparticles synthesized in benzyl alcohol: (a) overview, (b) higher magnification.

8 nm (light scattering). Representative transmission electron micrographs of the as-synthesized anatase nanoparticles are shown in Figure 2. An overview image at low magnification illustrates that the product is almost exclusively composed of small agglomerates of titania nanoparticles (Figure 2a). An image at higher magnification shows these agglomerates in more detail (Figure 2b). Although it is rather difficult to see the grain boundaries clearly, it can be said that the crystals are quite uniform in size and shape. According to Figure 2b, the average crystal size ranges from 2 to 5 nm, which is in good agreement with those calculated from XRD peak broadening (see below) but slightly smaller than the values obtained from light scattering. This discrepancy is likely due to the fact that the small agglomerates above persist in the liquid phase. However, one also has to keep in mind that light scattering measures the hydrodynamic radius, which is in general larger than the mere physical dimension, and its sensitivity drops at very small sizes (λ ) 633 nm). After preparation in benzyl alcohol, the slightly cloudy suspension was cooled down to room temperature and the product was recovered by precipitation in diethyl ether. The dried precipitate was ground into a white powder. X-ray diffraction clearly identifies the particles as highly crystalline anatase (Figure 3). No other crystalline titanium oxide polymorph seems to be present in combination with it. All the reflections are relatively broad due to the nanosize of the crystals. Scherrer analysis of the (101) peak points to an average crystallite size of about 4 nm. After their preparation, the nanoparticles are expected to feature a hydrophobic surface, covered by alkoxide groups. On the other hand, IR analysis shows absorptions in the OH stretching region (3000-3500 cm-1) as the main spectral feature, even for freshly precipitated material (Figure 4). A thermal treatment at 120 °C, causing desorption of any volatile compounds, has allowed assignment of the OH stretching absorption only partially to adsorbed water; while OH bending (1623 cm-1) substantially disappears (the remaining absorption in the region is centered at 1604 cm-1 and could be related to the aromatic ring breathing of benzyl groups), the permanence of a still strong absorption in the OH stretching area is an indication of nonvolatile OH groups, likely to identify as titanols. Noticeably, storage under air appears to produce no visible change in IR spectra.

Figure 4. ATR-IR spectra of nanoparticles after precipitation in diethyl ether before and after a thermal treatment at 120 °C for 1 h.

Thermogravimetric analysis (TGA) showed that the amount of adsorbed water (on nanoparticles kept under air for at least 12 h) corresponds to roughly 9% of the total sample weight (Figure 5, curve A). About 15% of the remaining dry weight (17% of the initial weight) is lost upon further heating; this could be ascribed both to loss of residual organic groups on the surface and to thermally activated condensation of titanols, to yield titanoxanes. It is known that exposure for times down to 1 h to temperatures as low as 300 °C can cause quantitative titanol condensation;38 however, this process is hardly rapid and quantitative at a lower temperature. If we therefore hypothesize the titanol condensation to correspond to the higher temperature part of the TGA curve (T ) 250-500 °C, above an inflection point present at about 250 °C), it would be possible to estimate the organic content to be about 6% of the dry weight (vs 11% of the water from condensation). Also in this case, storage does not apparently cause any change in the weight loss behavior (data not shown). Summary of Preparation. The nonaqueous acid decomposition of in situ produced titanium tetraethoxide in benzyl alcohol provides highly crystalline nanoparticles that feature rapidly hydrolyzable groups on the surface. Following workup, which (38) Kanta, A.; Sedev, R.; Ralston, J. Langmuir 2005, 21(6), 2400–2407.

Preparation of Ligand-Free TiO2

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Figure 5. Thermogravimetric curves (10 °C/min) of nanoparticles after precipitation in diethyl ether (A) and after redispersion in water (B). The weight loss likely caused by water desorption is calculated as the total weight loss after an isotherm 120 °C until constant weight; the differences between the two samples are very small; above all, the weight loss that can be ascribed to organic groups and water from titanol condensation (relative to the “dry” weight after water desorption) is substantially identical.

is performed under air and therefore allows contact with air moisture, the surface appears to be only marginally covered with organic residues, while it features large amounts of titanols and adsorbed water. We are inclined to think that the nanoparticle surface is originally covered by hydrolytically labile titanium ethoxide groups, which are rapidly cleaved by air moisture; benzyl groups, more stable, are likely present in smaller amounts (if at all). Nanoparticle Redispersion in a Water Environment. The white precipitate recovered from diethyl ether is easily redispersed in acidified water-ethanol (50:50 v/v) solution, resulting in a colorless and transparent suspension. The acid medium confers positive charge and therefore electrostatic repulsion and thus stability to the nanoparticles; without ethanol the precipitate redispersion is not complete and coarse aggregates can be seen, suggesting the presence of organic and hydrophobically associating groups. However, ethanol could be later removed by dialysis in acid-water, without producing any apparent change in particle size and charge (Figure 6). The redispersion process produced colloidal particles characterized by a large positive ζ potential, +44 mW at pH ) 1 (Figure 6A) and a size ranging between 10 and 100 nm, with an average value of 35 nm (Figure 6B) that represents a sound increase compared to their dimension before precipitation. Both TEM and AFM (Figure 7) confirm this finding, further showing the redispersed particles as aggregates of smaller objects, which can be identified as the original nanoparticles (Figure 7, inserts). Since neither ζ potential nor size appears to change during the solvent exchange and organic groups have substantially disappeared from IR spectra (Figure 8), it can be concluded that they have been completely hydrolyzed already in the hydroalcoholic solution. Since cluster formation cannot therefore be caused by hydrophobic association of surface groups, we are inclined to ascribe it to formation of interparticle hydrogen bonds between titanols following precipitation. Possibly this phenomenon can be minimized, for example by using a coprecipitant, i.e., a second, inert material soluble in the reaction environment but insoluble in ether, which would dilute the nanoparticles and thus reduce the likelihood of interparticle interactions. In any case, the aggregate dimensions are still perfectly acceptable for most applications in bionanomedicine.

Figure 6. Comparison of nanoparticles in hydroalcoholic solution (redispersion after precipitation) and in water (after dialysis). (A) ζ-potential distribution. The curves substantially overlap and the broader distribution in water-ethanol is most likely due to the presence of organic compounds (ethanol, traces of diethyl ether and benzyl alcohol) that affect the measurement. (B) Dynamic light scattering derived size distribution. The nanoparticles size does not appear to be affected by the change of solvent.

The final suspension was very stable at acidic pH (pH up to 4) where nanoparticles had a high ζ-potential value of +40-50 mV. With increase of pH, on the contrary, the ζ-potential decreases, with an apparent isoelectric point at pH 5-6; irreversible agglomeration, however, starts taking place before, at pH >4 (Figure 9). Interestingly, TGA clearly shows a substantial identity of the weight loss performance of the particles before and after redispersion in water (Figure 5). It is therefore possible to conclude that organic groups are still present after precipitation (IR) and can even complicate particle redispersion, but their overall amount is very small. A further analysis using XPS (see Supporting Information) provided the following results: (a) The Ti 2p band shows the typical splitting of Ti tetravalent atoms into two bands, a Ti 2p3/2 at 458.69 and a Ti 2p1/2 at 464.44 eV, due to spin-orbit interactions; these binding energies are consistent with literature data for titanium dioxide obtained by vapor deposition39 or alkoxide (hydrolytic) decomposition.40 (39) Tian, G. L.; Dong, L.; Wei, C. Y.; Huang, J. B.; He, H. B.; Shao, J. Opt. Mater. 2006, 28(8-9), 1058–1063. (40) Kumar, P. M.; Badrinarayanan, S.; Sastry, M. Thin Solid Films 2000, 358(1-2), 122–130.

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Figure 8. Comparison of IR spectra (recorded after a 10 min isothermal treatment at 120 °C) for nanoparticles precipitated in diethyl ether and redispersed (and dialysis) in acid-water. After precipitation, the presence of organic residues can be inferred by absorptions at 1604, 1564, 1495 (benzyl ring breathing), 1454 (δas CH3 and/or benzyl vibration), 1362 (possibly CH3 wagging, typical of TiOEt4), 1321 (unidentified), 1206 and 1071 cm-1 (possibly C-O stretching; TiOEt4 typically presents a double peak at 1050-1060 and 1100-1150 cm-1; partial hydrolysis and low pH may be the cause of the higher wavenumbers). The band at 1250-1260 can be associated to other C-O vibrations,29 possibly of benzyl groups.

Figure 7. (top) TEM image of TiO2 nanoparticles: it is apparent that individual nanoparticles have agglomerated into clusters. After dialysis specimens were prepared depositing a layer of methyl cellulose on the nanoparticles to avoid them to burst during the evacuation procedure (see Supporting Information); this phenomenon is likely due to capillary forces developing within the clusters at the interface between individual nanoparticles. (below) AFM image (amplitude mode, scan size 1 µm) of TiO2 particles deposited on mica from a water solution and dried in air. Cluster formation is evident here too, although the clusters appear larger than those in TEM (and DLS); we think this is related to the different drying process, which favors some further aggregation.

(b) The O 1s band shows two components at 530.13 (83.7%) and at 531.36 (12.3%), which are typically assigned to Ti4+-O and to O-H groups; however, a contribution of alcoholates’ C-O groups, which should fall at 531.7 eV, cannot be completely ruled out. A third, minor band is present at 532.5 eV, which could be related to small amounts of coordinatively unsaturated Ti atoms, which, however, are not seen in the Ti 2p band area (where they should appear with a binding energy of about 456.7 eV40). (c) Three peaks can be recognized in the C 1p area, although their overall intensity is very low, as an indication of the low

concentration of surface-carbon-containing species: the major band, at 285.25 eV (76.4%), is associated to aliphatic and aromatic C-C bonds. The first minor component, at 287.09 eV (15.1%), can be associated to C-O bonds; the ratio C-C to C-O bonds seems to suggest a larger presence of benzyl groups rather than ethyl residues, which is easily explained with a much lower reactivity of the first ones with water. A second minor component is recorded at 289.63 eV (8.6%; the peak, however, has an extremely low intensity), which would suggest the presence of carboxylic groups (which are not visible in IR spectra); however, considering also the very low intensity of this peak, its presence may also be caused by impurities. Summary of Redispersion in Water. The nanoparticles can be easily redispersed in acidic medium, although in an aggregated form that is likely held together by hydrogen bonds and polar interactions rather than hydrophobic association of organic groups. More detailed analyses show that organic residues substantially disappear (IR) during redispersion; however, since their loss is not really appreciated by TGA, most of the alkoxy groups were already hydrolyzed at the stage of precipitation and isolation. Surface Functionalization. Our principal target is the preparation of “naked” titania nanoparticles that are easily amenable to surface functionalization, in order to obtain more sophisticated and environmentally and biologically responsive nanoparticles for biomedical application.41 As a model for this functionalization, we have used an enediol ligand, dopamine; compounds of this class, e.g., ascorbic acid, catechols, and alizarins, are known to bind titanium oxide via a very stable, substantially covalent linkage.18–20 Following addition of dopamine, the nanoparticles are not appreciably modified in size. IR analysis indicates in a qualitative fashion the presence of adsorbed organic molecules as a result of the addition of dopamine to nanoparticles (Figure 10). However, (41) Tirelli, N. Curr. Opin. Colloid Interface Sci. 2006, 11(4), 210–216.

Preparation of Ligand-Free TiO2

Figure 9. ζ-Potential and average particle size of titania nanoparticles in water as a function of pH. The isoelectric point is recorded at pH ) 5.6, but flocculation already occurs at pH >4; the ζ-potential values recorded above this pH are therefore only indicative, since they are recorded on a fractionated sample.

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dopamine appears to be present in solution (Supporting Information, Figure 3) lets us conclude the color to be solely related to the formation of a chelate complex.19 A pure absorption spectrum of this species can be obtained by removing the scattering contribution to the total optical density (the original spectra are reported in Supporting Information); by plotting the absorbance at 435 nm (Figure 11), it is therefore possible to follow the formation of the complex as a function of the amount of added dopamine, in fact titrating the surface adsorption sites (Figure 12). Qualitatively similar curves are obtained by measuring through HPLC the residual dopamine concentration in solution after adsorption. It should be noted that both analytical methods may suffer significant experimental errors and therefore the following results have only a semiquantitative value: for HPLC sample manipulation (nanoparticles are removed by raising pH to neutrality and filtering the flocculated matter is critical), since dopamine can be adsorbed on the filter or be entrapped in the particle flocs, while a weak point for UV-vis could be the accuracy of baseline correction. The mechanism of catechol adsorption on titania has been long debated. A convincing contribution by Rodriguez has demonstrated the existence of two different adsorption equilibria,28 with dopamine units possibly present both as chelating agents with both hydroxyl groups onto one adsorption site (“monomolecular mechanism”) and as bridging groups between two neighboring sites (“bimolecular mechanism”).

{

Figure 10. ATR-IR spectrum of dopamine-coated nanoparticles (dopamine addition until saturation, see Figure 11, followed by dialysis to remove unbound dopamine), compared to those of ligand-free nanoparticles and dopamine. The presence of dopamine can be inferred from an absorption below 1500 cm-1, possibly related to a phenyl “breathing” vibration. Other absorptions appear as shoulders of titania peaks (dashed arrows). It is also apparent that the dopamine fingerprint region has dramatically changed, a finding confirmed by the extensive IR investigation of Gomez and co-workers.29

the resulting spectral features are sufficiently different from those of pure dopamine (possibly because of the changes in molecular charge and mass distribution following the chelate formation) to hinder a conclusive identification. A more unequivocal description of the adsorption process can be obtained by the optical properties of the suspensions, which develop a deep red-orange color instantaneously upon exposure to dopamine solutions. Although it could be argued this to arise also from solution oxidation of the ligand to o-quinone (most orthoquinones are red in color42), the fact that no oxidized (42) Berger, S.; Rieker, A. Identification and determination of quinones. In The Chemistry of Quinonoid Compounds; Patai, S., Ed.; John Wiley and Sons: London, 1974; Vol. 1, pp 163-229..

DA + tTi(OH) S tTiDA + H+ + H2O 2tTiOH + DA S tTi2DA(+2H2O)

(7)

Within this description, it appears from the above analytical formulation of the equilibria and from experimental results28 that the first mode of adsorption is not favored at acidic pH, where, on the contrary, the bimolecular mechanism should be predominant. The existence of two modes of adsorption has been confirmed through a thorough recent study,29 although the second mode of adsorption was associated not to dopamine bridging between two titanium atoms but to a molecularly adsorbed state of the ligand. Theoretical arguments suggest the possibility of even three different states (molecularly adsorbed, monodentate and bidentate complexation43). It must be noted that, whatever the products of the other adsorption mechanisms, only chelate complexes are supposedly associated to absorption in the visible range. By using a Langmuir model (see Experimental Section) we have attempted to qualitatively describe the adsorption mechanism on our titania nanoparticles, in order to possibly confirm the existence of multiple adsorption mechanisms, as it has been previously demonstrated on macroscopic substrates: (A) HPLC. Through eq 4 (see Experimental Section), it is possible to obtain both the volumetric concentration of any kind of available Ti sites, which for a nanoparticle concentration of 3.7 g/L and in a monomolecular adsorption mechanism is [Ti]0 ) 3.79 mmol/L corresponding 1.02 mmol/g of nanoparticles) and a Langmuir constant of more than 20000 L/mol (Table 1). (B) UV-vis. From the titration curves in Figure 12 (absorbance vs [DA]0) it is possible to extract two kinds of information: (1) the absorbance at saturation, i.e., if we would assume all surfacebound species to absorb in the same fashion, at high dopamine concentration one has (43) Vega-Arroyo, M.; LeBreton, P. R.; Rajh, T.; Zapol, P.; Curtiss, L. A. Chem. Phys. Lett. 2005, 406(4-6), 306–311.

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Figure 11. Addition of dopamine to a water suspension of titania nanoparticles at pH 1. (left) The UV-vis spectra of the suspension show a clear increase of optical density in the range of 400-500 nm due to the addition of titrant (from 0 to13 µmol of dopamine added to 2 mL of 0.37% w/w titania suspension). (right) The absorption at 435 nm (corrected by dilution) as an indication of the concentration of adsorbed dopamine. Please note that a scattering baseline has been subtracted from the spectra (the baseline is obtained from an exponential fitting of the parts of the spectrum which do not feature absorption bands), in order to distinguish absorbance increase from effects on aggregation or changes in refractive index. The original spectra and the noncorrected 435 nm optical density values are reported in the Supporting Information. Table 1. Summary of Data Calculated from Titration Curves analytical method, nanoparticle concn HPLC, 3.7 g/L UV-vis, 3.7 g/L UV-vis, 1.85 g/L UV-vis, 0.925 g/L

[Ti]0 (mM)

ε (L/mol)

KL (L/mol)

400 ( 10 350 ( 10 360 ( 15

21500 ( 3000 23800 ( 2500 1880 ( 150 970 ( 100

a

3.8 (7.78) 3.2 (7.4)a 2.2 (4.4)a 1.0 (2.0)a

a In parentheses the value calculated for a bimolecular adsorption mechanism.

Figure 12. Black symbols: absorbance at 435 nm as a function of dopamine total concentration ([DA]0) and nanoparticle concentration. White symbols: concentration of bound dopamine as a function of dopamine total concentration. Please note that these curves are not Langmuir isotherms since the x axis is not the free dopamine concentration at the equilibrium: they are “titration” curves.

lim (A) )

[DA]f∞

[Ti]0 x

where x ) 1 or 2 in the case of a monomolecular or bimolecular mechanism; (2) an equivalence point (the crossing point of the tangents at 0 and ∞) where [DA]0 ) [Ti]0/x. The two data points, combined, provide knowledge of both [Ti]0 and ε. While the [Ti]0 values substantially confirm that obtained from HPLC and are supposedly reliable, the real values of the extinction coefficients may be seriously underestimated, since [Ti]0 may comprise also nonadsorbing species; their usefulness is therefore in being “effective” extinction coefficients that allow linking the absorbance of chelates to the concentrations of all adsorbed species, although no information could be gathered on their relative quantities. Finally, knowing both [Ti]0 and ε, it is possible to rearrange eq 5 (see Experimental Section) in a form that allows easy linear fittings

(

)

(

1 1 x 1 x 1+ ) 1+ ) A [Ti]0 KL[DA]eq [Ti]0 KL[DA]0 - A ⁄ 

)

where again x ) 1 or 2. Although the corresponding graphs (Figure 13) show a reasonably linear behavior, the calculated

Figure 13. Plot of reciprocals of absorbance or bound dopamine concentration (from UV-vis, black symbols, or HPLC measurements, white symbols) vs reciprocal of free dopamine concentration. Langmuir isotherm fits as dotted lines.

Langmuir constants are markedly and apparently nonlinearly dependent on nanoparticle concentration (Table 1), which would support a predominantly bimolecular adsorption mechanism (dopamine as a bridging ligand). It is worth noting that for the most concentrated sample (3.7 g/L) both HPLC and UV-vis provide a KL value far larger than those of the two more diluted ones; one should keep in mind that these data are affected by large experimental errors: the optical density of the sample before subtraction of the scattering baseline is .1, while the loss of some dopamine during sample preparation for HPLC is unavoidable, and these factors may become critical when dealing with very low free dopamine concentrations, likely leading to an overestimation of KL.

Preparation of Ligand-Free TiO2

From the above findings we gather some qualitative indications: (A) the dependence of KL on nanoparticle concentration suggests the adsorption mechanism not to be predominantly based on chelate formation, which confirms literature hypotheses and experimental proofs on macroscopic (anatase) samples. (B) However, due to the development of a color, chelate complexes are surely formed, although possibly being a minor product. (C) The molecular mechanism of adsorption does not affect the amount of dopamine adsorbed per nanoparticle, which appears to be close to 1 mmol/g (0.15 g/g ) 15% wt).

Conclusions We have here demonstrated the feasibility to couple a nonaqueous preparative process (the acid-catalyzed condensation of in situ produced titanium tetraethoxide) with a surface functionalization in a water milieu. This method combines the high crystallinity obtainable with the nonaqueous process with a tailorable surface, allowing control of the two factors independently and in different times. Additionally, the surface groups can be also very polar, nonorganosoluble molecules that could not be employed in the nonaqueous synthetic process. Decoupling the preparative phase from the surface functionalization is advantageous in that it will allow libraries of identical, highly crystalline anatase nanoparticles differing only in surface composition; the introduction of surface groups during the preparative step would, on the other hand, produce differently sized nanoparticles depending on the ligand strength. Another

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key point is that the two-phase method allows a much finer control over the density of the functional surface groups or the use of different ligands with a precise knowledge of their final ratio(s). In perspective, these features are all beneficial for biomedical applications of titania nanocrystals, e.g., for photodynamic therapy, which would be boosted by the possibility of a flexible functionalization with targeting groups. As a final remark, it is noteworthy that, when we have used dopamine as a model enediol ligand, its adsorption mechanism appears not to predominantly imply chelate complexes, while interactions with two separate neighboring sites seem to be prevalent. Acknowledgment. NT is indebted to the Alexander von Humboldt foundation for a Bessel award which has allowed the initial phase of this work at the Max Planck Institute for Colloids and Interfaces NT wants also to thank the EPSRC for an Advanced Research Fellowship. The authors want to thank Prof. Markus Antonietti for all his support to the project. Supporting Information Available: Figures of XPS spectra of titania nanoparticles after precipitation in diethyl ether, TEM of titania nanoparticles deposited from aqueous acidic solution without a protective methyl cellulose layer, UV spectra of pure dopamine, of dopamine in the presence of titania nanoparticles, and of the filtrate of the dispersion, and UV-vis curves following addition of dopamine to titania nanoparticles. This material is available free of charge via the Internet at http://pubs.acs.org. LA800470E