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J. Phys. Chem. B 2002, 106, 3146-3150
Network Superstructure of Pseudoisocyanine J-Aggregates in Aqueous Sodium Chloride Solution Revealed by Cryo-Transmission Electron Microscopy Hans von Berlepsch* and Christoph Bo1 ttcher Forschungszentrum fu¨ r Elektronenmikroskopie der Freien UniVersita¨ t Berlin, Fabeckstrasse 36a, D-14195 Berlin, Germany ReceiVed: NoVember 29, 2001; In Final Form: January 23, 2002
Cryo-transmission electron microscopy (cryo-TEM) has been applied to characterize J-aggregates formed by the dye 1,1′-diethyl-2,2′-cyanine chloride (pseudoisocyanine chloride) in 200 mM sodium chloride solutions as a function of the dye concentration within the range of (2.5-6.1) × 10-4 mol/L. Electron micrographs reveal the formation of a network superstructure consisting of isolated fibers and complex fiber bundles. Upon dilution, more of the isolated threadlike J-aggregates become visible, which are characterized by a diameter of 2.3 nm, lengths of several hundreds of nanometers and a high stiffness. Such J-aggregates already appear at room temperature at a concentration as low as 2.5 × 10-4 mol/L. Ends of the threadlike J-aggregates can be found, but they are extremely rare, indicating a large end-cap energy. Because of their morphological appearance, the J-aggregates resemble ordinary polymers rather than “equilibrium polymers”. Concentrationdependent growth of J-aggregates cannot be proved. H-aggregates of mesoscopic size are not found.
Introduction Scheibe1
Jelly2
More than sixty years ago, and independently discovered that concentrated solutions of the dye 1,1′-diethyl2,2′-cyanine chloride (pseudoisocyanine chloride, PIC-Cl)
develop a narrow and, with respect to the monomer absorption spectrum, red-shifted absorption band, now commonly referred to as the J-band. Scheibe attributed this peculiar spectroscopic behavior to a reversible polymerization of the chromophores due to intermolecular interactions into threadlike aggregates.3 Later, many other cyanine and merocyanine dyes were found that show the same aggregation phenomenon. Already in 1938, Franck and Teller4 adopted a Frenkel-type exciton approach to explain the spectral properties of J-aggregates, which is now universally accepted.5 Because of their giant absorption cross section and the ability of fast and efficient excitation energy migration over many molecules,6,7 supramolecular dye assemblies have recently become popular model systems to mimic light-harvesting complexes of photosynthetic bacteria and plants.8,9 Despite the multitude of studies and data available on optical and spectroscopic properties of PIC and related dyes, the knowledge of the supramolecular structure of the aggregates is still fragmentary and an object of scientific activity.10-16 In contrast to crystalline PIC,17,18 the exact packing structure of individual PIC molecules within the extended linear J-aggregates is not clear up to now. Recently, we could directly visualize for the first time the rodlike morphology of the PIC-Cl J-aggregates in aqueous solution.19 Using cryo-transmission * To whom correspondence should be addressed.
electron microscopy (cryo-TEM), we estimated a rod diameter of 2.3 nm and proposed alternative structure models for the molecular packing within the aggregate. Since the early studies of Scheibe, it is known that PIC-Cl solutions exhibit strong viscoelasticity and form gels above a dye concentration of about 1 × 10-2 mol/L. Compared to viscoelastic solutions of elongated micelles, which represent another self-aggregation system, the respective critical dye concentration is rather low. Rehage et al.20 have studied the rheological properties of PIC-Cl solutions in detail. Their data indicated (i) the presence of supermolecular network structures or (ii) the shear-induced deformation and orientation of rod-shaped aggregates. Our recent cryo-TEM19 study indeed proved the existence of a homogeneous and closely packed network of rodlike aggregates at 12.5 × 10-3 mol/L. Rehage et al. studied also the dynamic properties and did not find a monoexponential stress relaxation as commonly observed for viscoelastic surfactant solutions. Instead, the distribution of relaxation times was rather broad reminiscent of the dynamic properties of entangled polymer solutions. Also, the scaling behavior of the plateau modulus as a function of the dye concentration was in fairly good agreement with that of entangled polymers. These findings raised several questions about the analogy of viscoelastic dye and surfactant solutions, which will be addressed in this study. One would expect that dye aggregates because of their selfaggregating nature grow in length with concentration, as surfactant micelles do. The extremely strong increase of the J-band’s absorbance with concentration in the relatively narrow concentration range between 1 × 10-2 and 1 × 10-3 mol/L could indicate such rapid growth. Another possible explanation might be that the strong increase of absorbance is solely due to the increasing concentration of aggregates with more or less fixed length distribution. The dynamic properties of the dye aggregates are obviously not mainly controlled by scission and recombination reactions (average lifetime of the aggregates) as
10.1021/jp0143701 CCC: $22.00 © 2002 American Chemical Society Published on Web 03/05/2002
Network Superstructure of Pseudoisocyanine in the case of elongated micelles21 but are rather determined by the diffusion time of the entire aggregate. H-aggregates are spectroscopic entities that are characterized by a blue-shifted absorption band with respect to monomer absorption. H-aggregates appear at low concentration prior to the formation of J-aggregates. Until now, their physical size and structure were controversially discussed. It has not been clarified experimentally whether they really represent dimers, as commonly assumed,22 or whether they represent particles of mesoscopic size, which upon increase of the concentration further assemble to form eventually J-aggregates.16 The present cryo-TEM study was aimed to answer some of these questions. Cryo-TEM studies can provide direct images of dye aggregates in their native environment down to the nanometer scale, as has been demonstrated in our recent studies on different dye systems.23,24 In contrast to scattering methods, the interpretation of the data is not model-dependent. In particular, interparticle interactions and corrections taking into account the residual amount of unaggregated dye16 need not be considered. However, a special problem arises for the highly water-soluble PIC-Cl from the rather large threshold concentration on the order of 5 × 10-3 mol/L at which J-aggregation starts. At 12.5 × 10-3 mol/L, the J-aggregates forming the network are so closely packed, that single isolated J-aggregates cannot be studied. Thus, for the present purpose, dilution was a strict requirement. Because dilution was expected to affect aggregation, we chose to work in salt solution proceeding on the well-known finding that addition of inorganic salts enhances J-aggregation in PIC solutions.25,26 By adding 200 mM sodium chloride, the threshold concentration could be reduced by about 1 order of magnitude. This turned out to be sufficient for visualizing isolated aggregates, as the reported results will show. Experimental Section PIC-Cl was obtained as a gift from AGFA AG and was used without further purification. The dye solutions were prepared in Milli-Q water containing 0.2 mol/L sodium chloride by stirring under gentle heat. NaCl (p.a.) was purchased from Merck. The absorption spectra were measured with a Varian Cary 4 spectrophotometer in quartz cells of 100 µm optical path length. All spectroscopic measurements were carried out at room temperature (21 °C). The samples for cryo-TEM were prepared at room temperature by placing a droplet (10 µL) of a fresh solution on hydrophilized perforated carbon-filmed grids (60 s plasma treatment at 8 W using a BALTEC MED 020 device). The excess fluid was blotted off to create an ultrathin layer (typical thickness of 100 nm) of the solution spanning the holes of the carbon film. The grids were immediately vitrified in liquid ethane at its freezing point (-184 °C) using a standard plunging device. Ultrafast cooling is necessary for an artifact-free thermal fixation (vitrification) of the aqueous solution avoiding crystallization of the solvent or rearrangement of the assemblies. The vitrified samples were transferred under liquid nitrogen into a Philips CM12 transmission electron microscope using the Gatan cryoholder and -stage (model 626). Microscopy was carried out at -175 °C sample temperature using the microscopes’ lowdose protocol at a primary magnification of 58 300×. The defocus was chosen to be 1.2 µm corresponding to a first zero of the phase contrast transfer function at 2.1 nm. Results and Discussion Prior to the cryo-TEM investigations, the absorption spectra of the solutions were thoroughly investigated to discriminate
J. Phys. Chem. B, Vol. 106, No. 12, 2002 3147
Figure 1. Set of absorption spectra of PIC-Cl solutions in 200 mM NaCl at various concentrations, cD, measured at 21 °C in cells of 100 µm optical path length (solid lines). Dye concentration (in mol/L) from top to button is as follows: 6.0 × 10-4, 4.1 × 10-4, 3.1 × 10-4, 2.5 × 10-4. The ordinate was cut to enlarge the short-wavelength part of the spectra. The extinction of the cut J-band (maximum at 573 nm) of the 6.0 × 10-4 mol/L solution was 1.3. The normalized spectrum of the monomer solution (cD ) 2.35 × 10-5 mol/L) in water has been added (dotted curve).
between monomers and different aggregate species. Figure 1 shows a set of absorption spectra for various PIC-Cl concentrations, cD, in 200 mM NaCl solution taken at room temperature. The monomer spectrum measured in water at highly dilute concentration is added to the figure. For the highest dye concentration of 6.0 × 10-4 mol/L, a spectrum with the typical narrow J-band peaked at ∼573 nm and two further maxima at ∼494 and ∼525 nm is found. All of these peaks appear upon J-aggregation.3,27,28 The band with a maximum at around 494 nm is polarized perpendicular to the aggregate axis, as dichroic measurements on oriented samples have shown.3,19,29 The monomer spectrum is characterized by the main transition (0,0 transition) at ∼523 nm and by at least two vibrational satellites, the first one located around 490 nm. Upon decreasing the dye concentration, the position of the J-band (573 nm) remains fixed, while its extinction value drastically decreases. It is important to note that the J-band is still visible down to cD ) 2.5 × 10-4 mol/L, that is, J-aggregates should still exist at this low concentration. The absorbance of the two other bands also decreases upon dilution, but in addition, they slightly shift to smaller wavelengths. H-aggregates are known to have transitions at ∼482 and ∼523 nm.22 The occurring shift of the shortest wavelength band to about 485 nm (for the lowest concentration of 2.5 × 10-4 mol/ L) thus obviously arises from an increased amount of Haggregates at the expense of a decreasing contribution from J-aggregates. The band around 525 nm is obviously due to a superposition of monomers, H-, and J-aggregates, which all have transitions there. If absorption spectra obtained at low concentration in pure water (cf., for example, ref 3 or our recent paper19) are compared to those in the presence of salt, one can notice marked differences in the intensity of the two shortwavelength bands. The absorbance at ∼485 nm is usually larger than at ∼525 nm. This finding obviously means that the amount of H-aggregates is larger in the salt-free solution, or equivalently, the amount of monomers is larger in the presence of salt.16 Figure 2a shows a typical cryo-TEM image of the 6.0 × 10-4 mol/L solution. A loose network of fibers of about 10 nm thickness is found, whereby the mesh size of this network is on the order of 100 nm. These fibers are bundles consisting of interwoven single threadlike J-aggregates, of which the diameter
3148 J. Phys. Chem. B, Vol. 106, No. 12, 2002
von Berlepsch and Bo¨ttcher
Figure 2. Cryo-TEM images of PIC-Cl solutions taken at different dye concentrations in the presence of 200 mM NaCl (bar ) 50 nm): (a) network of fiber bundles embedded in vitreous ice covering a hole of the supporting carbon film at 6.0 × 10-4 mol/L; (b) bundles of fibers as well as single J-aggregates at 4.1 × 10-4 mol/L; (c) occasionally, ends of single J-aggregates can be identified (arrows) (cD ) 3.1 × 10-4 mol/L); (d) single J-aggregate at 2.5 × 10-4 mol/L.
of 2.3 ( 0.2 nm agrees well with that obtained for the J-aggregate in the absence of salt and of which the persistence length is at least on the order of 100 nm. Thin isolated J-aggregates fill up the meshes of the coarse-meshed network consisting of the closely packed fiber bundles. The network morphology differs strongly from that described in our recent study in the absence of salt at a dye concentration of 12.5 ×
10-3 mol/L. No bundle formation has been detected but a homogeneous network of single strands. However, a very similar morphology has been observed for spin-coated thin films of PIC-J in poly(vinyl sulfate) by Barbara et al.11 The network formation is obviously the reason for the appreciable viscosity of the solutions. A gellike state has been found in 200 mM NaCl solution at a concentration as low as 1 × 10-3 mol/L.
Network Superstructure of Pseudoisocyanine The compactness of the network decreases with decreasing dye concentration as is demonstrated in Figure 2b,c for cD ) 4.1 × 10-4 and 3.1 × 10-4 mol/L, respectively. Fiber bundles are still present. Some of them are rather compact and twisted, while others appear to be only loose and nearly parallel arrangements of single strands. The latter could be an ordering effect upon preparation of very thin layers (100 nm) for vitrification. To explain the formation of the more compact bundles, attractive forces between single strands have to be assumed. Attractive forces between like-charged macromolecules have recently been the subject of series of experiments and theoretical studies (cf. ref 30 and further references given there). Here, the effect is due to small amounts of polyvalent counterions condensed on the polyelectrolytes. In our present case of PIC-Cl J-aggregates, the high amount of chloride counterions will screen the strong electrostatic repulsion between the single strands. The Debye screening length in 200 mM NaCl is about 0.7 nm, and attractive forces such as hydrophobic interactions could have become operative. It is known for a long time25 that J-aggregation of PIC-Cl in salt solution strongly depends on the anion of the added inorganic salt. Thus, one might conclude, that counterionspecific interactions might also be important. The exact mechanism, however, leading to bundle formation is not clear at present, and further work is necessary for explanation. To interpret the unusual rheological behavior, it is interesting to know whether, besides the formation of bundles and entanglements, other cross-linking mechanisms also occur. However, on inspecting a multitude of images, so-called “Y junctions”31 have not been found. Thus, we believe that true branching points between the J-aggregates do not exist. Indeed, because of the anisotropic nature of the molecular packing within the PIC J-aggregate19 a strongly curved packing at the seam between two branches appears energetically highly unfavorable. Single J-aggregates are generally many hundreds of nanometers long and ends are very rarely found. Two rare examples of ends are visible in Figure 2c (indicated by arrows). With an estimated number of about 8.3 molecules per strand and nanometer,19 aggregation numbers reaching from 1000 to 10 000 may be deduced. In terms of the ladder model,32 which has proven to be a successful thermodynamic description of surfactant self-aggregation, the nearly complete absence of ends corresponds to a much larger end-cap energy as compared to ordinary surfactant systems.33 The end-cap energy is the energy required to create two chain ends (or to break a chain into two parts). The average length of neutral or screened ionic micelles, 〈L〉, depends on the surfactant concentration, c, and the endcap energy, Ec, according to the relation 〈L〉 ∝ c1/2 exp(Ec/ (2kBT)),34 where kBT is the thermal energy.34 Under the experimental conditions used (i.e., temperature, ionic strength) and within the investigated range of concentrations, there is no evidence for noticeable growth or a limited average lifetime of the J-aggregates, respectively. In particular, at the lowest concentration of 2.5 × 10-4 mol/L, cf. Figure 2d, where even J-aggregates are very rarely found, the aggregates appear practically endless. From their morphology, the J-aggregates thus resemble ordinary polymers with fixed (quenched) length distribution rather than “equilibrium polymers”. This finding is in agreement with the observation of Rehage et al.20 that saltfree PIC-Cl solutions exhibit a dynamical rheological behavior as entangled polymer solutions. Although both systems differ in composition and concentration, the good agreement is surprising and indicates a general tendency of the PIC-Cl dye to form quasi-endless J-aggregates in the beginning of aggregation. The initially rapid aggregation could not be resolved, and
J. Phys. Chem. B, Vol. 106, No. 12, 2002 3149 the sudden occurrence of the J-band within the narrow concentration range (from 2.5 × 10-4 to 6 × 10-4 mol/L) seems to be mainly the result of an increasing concentration of J-aggregates and not due to their linear growth. The physical reason is obviously the extremely large end-cap energy. It appears plausible that the highly anisotropic nature of the sandwich-like packing of dye molecules within the J-aggregate19 results in high free energy costs for a differing packing. Indeed, spherical packing of molecules as in case of the hemispherical end-caps terminating cylindrical micelles seems energetically unfavorable for dye molecules. The formation of closed-loop J-aggregates seems unlikely too because of the high stiffness of aggregates. Returning to the cryo-TEM image for the lowest concentration (Figure 2d) we would like to emphasize that with the exception of rarely occurring J-aggregates no other particles can be found. Dye monomers or dimers were not expected to be visible due to their smallness and low contrast. As derived from crystallographic data, the length of the PIC molecule is approximately 1 nm. Particles of mesoscopic size, having an aggregation number on the order of 55 as observed by light scattering and identified by Neumann16 as H-aggregates, should in principle be visible but have not been found. Thus, we have no experimental indication of H-aggregates being larger than a few molecules. Conclusions Cryo-TEM investigations on PIC-Cl solutions with dye concentrations ranging from 2.5 × 10-4 to 6.1 × 10-4 mol/L in 200 mM NaCl revealed the formation of a network superstructure, which explains the high viscoelasticity of the solutions. The strands forming the network are bundles of J-aggregates as well as single J-aggregates of 2.3 nm thickness, several hundreds of nanometer length and high stiffness. The bundle formation at high ionic strength could be due to attractive hydrophobic forces or counterion interaction effects. Upon dilution, single isolated threadlike J-aggregates become visible. They already appear at room temperature at a concentration as low as 2.5 × 10-4 mol/L. Ends can be found but are extremely rare, indicating a large end-cap energy of the J-aggregates. Because of their morphological appearance the J-aggregates resemble ordinary polymers rather than “equilibrium polymers”. This could explain the recently observed polymer-like rheological behavior of aggregated PIC-Cl solutions. Concentrationdependent growth of J-aggregates cannot be proved. The sudden occurrence of the J-band within the narrow concentration range from 2.5 × 10-4 to 6 × 10-4 mol/L seems to be mainly the result of an increasing concentration of aggregates and not due to their growth. No further particles of mesoscopic size, in particular H-aggregates, could be detected, supporting the established view of their small size. Acknowledgment. Financial support by the Deutsche Forschungsgemeinschaft (SFB 448 “Mesoskopisch strukturierte Verbundsysteme”) is gratefully acknowledged. References and Notes (1) Scheibe, G. Angew. Chem. 1936, 49, 563; Angew. Chem. 1937, 50, 51. (2) Jelly, E. E. Nature 1936, 138, 1009; Nature 1937, 139, 631. (3) Scheibe, G. In Optische Anregung Organischer Systeme; Foerst, W., Ed.; Verlag Chemie: Weinheim, Germany, 1966; p 109. (4) Franck, J.; Teller, E. J. Chem. Phys. 1938, 6, 861. (5) Kobayashi, T., Ed. J-Aggregates; World Scientific: Singapore, 1996.
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