To investigate diffusion processes in agarose gel, nanoparticles with sizes in the range between 1 and 140 nm have been tested by means of fluorescence correlation spectroscopy. Understanding the diffusion properties in agarose gels is interesting, because such gels are good models for microbial biofilms and cells cytoplasm. The fluorescence correlation spectroscopy technique is very useful for such investigations due to its high sensitivity and selectivity, its excellent spatial resolution compared to the pore size of the gel, and its ability to probe a wide range of sizes of diffusing nanoparticles. The largest hydrodynamic radius (R(c)) of trapped particles that displayed local mobility was estimated to be 70 nm for a 1.5% agarose gel. The results showed that diffusion of particles in agarose gel is anomalous, with a diverging fractal dimension of diffusion when the large particles become entrapped in the pores of the gel. The latter situation occurs when the reduced size (R(A)/R(c)) of the diffusing particle, A, is >0.4. Variations of the fractal exponent of diffusion (d(w)) with the reduced particle size were in agreement with three-dimensional Monte Carlo simulations in porous media. Nonetheless, a systematic offset of d(w) was observed in real systems and was attributed to weak nonelastic interactions between the diffusing particles and polymer fibers, which was not considered in the Monte Carlo simulations.

Fluorescence correlation spectroscopy (FCS) was used to determine diffusion coefficients (D) of the Suwannee River fulvic and humic acids (SRFA and SRHA) and to measure the effects of pH, ionic strength, Ca, and humic substance (HS) concentration on the value of D. For these HS, average diffusion coefficients were in the range of (2−3) × 10-10 m2 s-1 (corresponding to hydrodynamic diameters of approximately 1.5−2.1 nm). Small, but significant, decreases in the diffusion coefficients were observed with decreasing pH, most likely indicating that a small degree of aggregation (formation of dimers and trimers) was occurring. The effect of ionic strength (up to 100 mM) was either small or insignificant, but, where a change occurred, there was a tendency toward a reduction in the diffusion coefficient with increasing salt concentration. No effect of HS concentration (1−50 mg L-1) or hydration time (1−14 days) was observed. There were no observable effects of Ca in comparison to Na, at an ionic strength of 5 mM. For all conditions studied, SRFA had a higher diffusion coefficient than SRHA which is in agreement with literature data on their respective molar masses. Since this is the first application of FCS to HS, the technique was systematically tested for artifacts, in particular photo bleaching and the effect of fluorescence excitation wavelength. Despite a small amount of photobleaching due to the intense laser excitation, FCS was found to be suitable for use with fulvic or humic acids.

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Measurements of the diffusion coefficients of five different humic substances (HS) have been performed in water and in agarose hydrogels at several pH values (in the range of 3-10) and gel concentrations (in the range of 0.7-3% w/w). Fluorescence correlation spectroscopy (FCS) and classical diffusion cells were used in parallel to probe diffusion over both microscopic and mesoscopic distance scales. In general, agreement between the techniques was reasonable, which indicated that local nonhomogenities in the gel did not play an important role. Diffusion coefficients (D) in the gel were generally in the range of 0.9-2.5 x 10(-10) m2 s(-1) but were generally only 10-20% lower than in solution. At low pH values, one of the studied humic substances (a peat humic acid, PPHA) formed large aggregates that could not penetrate into the gel and therefore could not be defined by a single D value. The observed decreases of D in the gel for other HS were too large to be explained by the tortuousity and obstructive effects of the gel alone. D decreased slightly with increasing gel concentration and increased slightly with pH. Because modifications of D due to pH were similar in both the gel and the free solution, it is unlikely that complexation with the gel was greatly influenced by the pH. Rather, the main effect that appeared to decrease the diffusive flux in gels was likely small increases in the hydrodynamic radii of the humic macromolecules. An anomalous diffusion model was used to describe the FCS data in the gel. The characteristic exponent determined by fitting the autocorrelation functions with this model decreased only slightly (from 0.96 to 0.90) with increasing gel concentration providing support that HS complexation with the gel fibers was not very important. The results have important implications for our understanding of the fate and behavior of the HS and their associated pollutants and for interpreting metal speciation data obtained using gel-covered analytical sensors.

In the present work, we have studied the adsorption of Rhodamine 6G on negative colloidal particles (Silica-Bindzil, Ludox-SM30 and Ludox-HS30) and Calcein on positive colloidal particles (Ludox-CL and Alumina) at very low concentrations by use of fluorescent correlation spectroscopy (FCS). In this relation we have introduced a set of equations which take into account the different quantum yield of the dye molecules and the particles as well as the adsorption of the dye molecules on the sample cell. The concentrations of fluorescent dye were varied between 2 × 10 -9 and 2 × 10 -7 M, and the concentrations of particles were in the range of 0.01-0.03 g/L. The adsorption data fit well with a Langmuir adsorption isotherm. The values of the adsorption constants at pH 5.5-5.7, ionic strength NaCl 1 mM, and room temperature (22 ( 0.5 °C) were in the range (0.2-2.5) × 10 8 L/mol, and the adsorption energy varied between -10 and 13 kJ/mol. The cooperative binding model (generalized Freundlich) was also checked but showed less satisfactory results. This work demonstrates the high performance of the FCS method for investigation of the adsorption equilibrium at solid-liquid interface. In particular the technique does not required any separation step of the adsorbed and nonadsorbed species and is not influenced by the adsorption on the sample cell walls which is often a source of artifacts when working with very low concentrations.

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The motion of fluorescent latex beads and circular DNA in agarose gels is investigated by epifluorescence computer-enhanced microscopy. The individual trajectories are obtained and the diffusion dynamics are determined by averaging over an ensemble of trajectories. It was found that the diffusion of latex beads in agarose gels is characterized by the scaling relation ( )2 ∼ t 2/ d W with d W = 3.1 ± 0.3, in the gel concentration range 0.6%−1.2%. This indicates that the structure of the holes in agarose gels is fractal on the scale from 0.2 to 15 μm that has been studied and different from those of percolating clusters. If an electric field is applied, d W measured in the direction of the field decreases and can even reach values lower than 2; this means that the diffusion increases at long time. The mobility vs gel concentration graph is found to be well fitted with a polynomial expression in agreement with recent computer calculations. Finally our preliminary results on DNA show that the motion of M13 circular DNA molecules is governed by the same dependencies as those of the latex particles.