Free Diffusion Of Solutes Research Articles

Charge-Gated Transport of Proteins in Nanostructured Optical Films of Mesoporous Silica

The admission into and diffusion through nanoscale pores by molecules is a fundamental process of great importance to biology and separations science. Systems (e.g., chromatography, electrophoresis) designed to harness such processes tend to remove the separation process from the detection event, both spatially and temporally. Here, we describe the preparation and characterization of thin optical Fabry-Pérot films of mesoporous silica (pSiO(2)) that can detect protein infiltration by optical interferometry, which probes the separation process in real time and in the same ultrasmall physical volume (5 nL). Admission of a protein into the pores is controlled by the diameter (∼50 nm) and the surface charge of the pores, and both the rate and the degree of protein infiltration are a function of solution pH. Test proteins bovine serum albumin (BSA, 66 kDa), bovine hemoglobin (BHb, 65 kDa), and equine myoglobin (EMb, 18 kDa) are admitted to or excluded from the nanophase pores of this material based on their size and charge. The rate of protein transport within the pores of the pSiO(2) film is slowed by 3 orders of magnitude relative to the free-solution diffusion values, and it is maximized when pH = pI.

Adsorption of deamidated antibody variants on macroporous and dextran-grafted cation exchangers: II. Adsorption kinetics

Single and multicomponent batch adsorption kinetics were obtained for deamidated mAb variants on two commercial cation exchangers, one with an open macroporous structure – UNOsphere S – and the other with charged dextran grafts – Capto S. The adsorption kinetics for the macroporous matrix was found to be controlled largely by pore diffusion. The effective diffusivity estimated from single component data was a fraction of the mAb free solution diffusivity, and its value could be used to accurately predict the adsorption kinetics for two- and three-component systems. In this case, when two or more variants were adsorbed simultaneously, both experimental and predicted results showed a temporary overshoot of the amount adsorbed above the equilibrium value for the more deamidated variant followed by a gradual approach to equilibrium. Adsorption rates on the dextran grafted material were much faster than those observed for the macroporous matrix for both single component and simultaneous adsorption cases. In this case, no significant overshoot was observed for the more deamidated forms. The Capto S adsorption kinetics could be described well by a diffusion model with an adsorbed phase driving force for single component adsorption and for the simultaneous adsorption of multiple variants. However, this model failed to predict the adsorption kinetics when more deamidated forms pre-adsorbed on the resin were displaced by less deamidated ones. In this case, the kinetics of the displacement process was much slower indicating that the pre-adsorbed components severely hindered transport of the more strongly bound variants. Overall, the results indicate that despite the lower capacity, the macroporous resin may be more efficient in process applications where displacement of one variant by another takes place as a result of the faster and more predictable kinetics.

Dextran-grafted cation exchanger based on superporous agarose gel: Adsorption isotherms, uptake kinetics and dynamic protein adsorption performance

A novel chromatographic medium for high-capacity protein adsorption was fabricated by grafting dextran (40 kDa) onto the pore surfaces of superporous agarose (SA) beads. The bead was denoted as D-SA. D-SA, SA and homogeneous agarose (HA) beads were modified with sulfopropyl (SP) group to prepare cation exchangers, and the adsorption and uptake of lysozyme on all three cation-exchange chromatographic beads (SP-HA, SP-SA and SP-D-SA) were investigated at salt concentrations of 6–50 mmol/L. Static adsorption experiments showed that the adsorption capacity of SP-D-SA (2.24 mmol/g) was 78% higher than that of SP-SA (1.26 mmol/g) and 54% higher than that of SP-HA (1.45 mmol/g) at a salt concentration of 6 mmol/L. Moreover, salt concentration had less influence on the adsorption capacity and dissociation constant of SP-D-SA than it did on SP-HA, suggesting that dextran-grafted superporous bead is a more potent architecture for chromatographic beads. In the dynamic uptake of lysozyme to the three cation-exchange beads, the D e/ D 0 (the ratio of effective pore diffusivity to free solution diffusivity) values of 1.6–2.0 were obtained in SA-D-SA, indicating that effective pore diffusivities of SP-D-SA were about two times higher than free solution diffusivity for lysozyme. At 6 mmol/L NaCl, the D e value in SA-D-SA (22.0 × 10 −11 m 2/s) was 14.4-fold greater than that in SP-HA. Due to the superior uptake kinetics in SA-D-SA, the highest dynamic binding capacity (DBC) and adsorption efficiency (the ratio of DBC to static adsorption capacity) was likewise found in SP-D-SA. It is thus confirmed that SP-D-SA has combined the advantages of superporous matrix structure and drafted ligand chemistry in mass transport and offers a new opportunity for the development of high-performance protein chromatography.

Role of Surface Reorganization on Preferential Adsorption of Macromolecular Ensembles at the Solid/Fluid Interface

The adsorption of micelles made from precisely synthesized branched block copolymers is investigated and analyzed using a model framework that incorporates the effects of mass transport and dynamic relaxation/reorganization events occurring at the solid/fluid interface. Both processes are required to represent adequately the adsorption profile over the entire progression to pseudoequilibrium. Insight into the relative importance of the two processes, the terminus of the diffusion-dominated regime, and differences between diffusion in free solution and in confinement is also provided. The results demonstrate commonality between adsorption of micelle-forming surfactant-like copolymers and biomimetic vesicles formed by small-molecule surfactants, both of which are systems dominated by rearrangements on the surface.

Protein adsorption kinetics in charged agarose gels: Effect of agarose content and modeling

AbstractThe adsorption kinetics of myoglobin in charged gels of varying agarose content have been measured macroscopically, through batch uptake experiments, and microscopically, using light microscopy with gels supported in microfluidics chips. The apparent effective pore diffusivities, determined by fitting either set of rate data to the shrinking core model, were greater than the free solution diffusivity and concentration‐dependent. Moreover, the microscopically derived concentration profiles were qualitatively different from the predicted ones. Therefore, a new model taking into account an assumed favorable partitioning of the protein in the pore liquid is proposed to describe the adsorption kinetics. The new model yields effective pore diffusivities that are in approximate agreement with the values determined chromatographically under nonbinding conditions and with hindered diffusion theory. In addition, it predicts concentration profiles in the gel that are consistent with those observed microscopically. The overall increase in mass transfer is attributed to the favorable partitioning of the protein in the pores at low ionic strength, which results in a greater diffusional driving force. © 2008 American Institute of Chemical Engineers AIChE J, 2009

Rapid monoclonal antibody adsorption on dextran-grafted agarose media for ion-exchange chromatography

The binding capacity and adsorption kinetics of a monoclonal antibody (mAb) are measured for experimental cation exchangers obtained by grafting dextran polymers to agarose beads and compared with measurements for two commercial agarose-based cation exchangers with and without dextran grafts. Introduction of charged dextran polymers results in enhanced adsorption kinetics despite a dramatic reduction of the accessible pore size as determined by inverse size-exclusion chromatography. Incorporation of neutral dextran polymers in a charged agarose bead results instead in substantially lower binding capacities. The effective pore diffusivities obtained from batch uptake curves increase substantially as the protein concentration is reduced for the resins containing charged dextran grafts, but are much less dependent on protein concentration for the resins with no dextran or uncharged dextran grafts. The batch uptake results are corroborated by microscopic observations of transient adsorption in individual particles. In all cases studied, the adsorption kinetics is characterized by a sharp adsorption front consistent with a shell-progressive, diffusion limited mechanism. Greatly enhanced transport rates are obtained with an experimental resin containing charged dextran grafts with effective pore diffusivities that are 1–9 times larger than the free solution diffusivity and adsorption capacity approaching 300 mg/cm 3 of particle volume.

Dilution potential (DP) across rat alveolar epithelial cell monolayers

In this study, we estimated permeability ratio (PNa/PCl) between Na and Cl for paracellular pathways of primary rat alveolar epithelial cell monolayers (RAECM). PNa/PCl was estimated using Goldman‐Hodgkin‐Katz equation and DP measured across RAECM at 5–7 days. To inhibit Na pump activities, RAECM were bathed on both sides with K‐free Ringer's solution (KFRS, in which 5 mM KCl is replaced by equimolar NaCl). DP was measured after lowering apical [NaCl] tenfold by replacing apical fluid with a 9:1 mixture of isotonic K‐free mannitol solution and KFRS. Osmolarity of all solutions was adjusted to 300 mOsm with mannitol. DP was further corrected for liquid junction potential of voltage‐sensing electrodes across blank filters. Results show that equivalent short‐circuit current across RAECM bathed on both sides with KFRS is not significantly different from zero with electrical resistance >1,000 Ωcm2. When apical [NaCl] was lowered tenfold, corrected DP ∼ −13 mV (apical as reference) was observed, yielding PNa/PCl ∼1.8. For comparison, the free solution diffusivity ratio of Na to Cl is ∼0.7, while paracellular PNa/PCl of various epithelial barriers ranges from ∼0.2 and ∼0.5 for frog skin and bullfrog lung to ∼1.3 and ∼10 for rabbit proximal tubule and rat jejunum, respectively. These data indicate that passive ion conductance through tight junctions of RAECM is greater for Na than Cl. (Support: NIH and Hastings Foundation.)

Kinetic Model for Ca2+-induced Permeability Transition in Energized Liver Mitochondria Discriminates between Inhibitor Mechanisms

Cytotoxicity associated with pathophysiological Ca(2+) overload (e.g. in stroke) appears mediated by an event termed the mitochondrial permeability transition (mPT). We built and solved a kinetic model of the mPT in populations of isolated rat liver mitochondria that quantitatively describes Ca(2+)-induced mPT as a two-step sequence of pre-swelling induction followed by Ca(2+)-driven, positive feedback, autocatalytic propagation. The model was formulated as two differential equations, each directly related to experimental parameters (Ca(2+) flux/mitochondrial swelling). These parameters were simultaneously assessed using a spectroscopic approach to monitor multiple mitochondrial properties. The derived kinetic model correctly identifies a correlation between initial Ca(2+) concentration and delay interval prior to mPT induction. Within the model's framework, Ru-360 (a ruthenium complex) and Mg(2+) were shown to compete with the Ca(2+)-stimulated initiation phase of mPT induction, consistent with known inhibition at the phenomenological level of the Ca(2+) uniporter. The model further reveals that Mg(2+), but not Ru-360, inhibits Ca(2+)-induced effects on a downstream stage of mPT induction at a site distinct from the uniporter. The analytical approach was then applied to promethazine, an FDA-approved drug previously shown to inhibit both mPT and ischemia-reperfusion injury. Kinetic analysis revealed that promethazine delayed mPT induction in a manner qualitatively distinct from that of lower concentrations of Mg(2+). In summary, we have developed a kinetic model to aid in the quantitative characterization of mPT induction. This model is consistent with/informative about the biochemistry of several mPT inhibitors, and its success suggests that this kinetic approach can aid in the classification of agents or targets that modulate mPT induction.

Protein adsorption and transport in agarose and dextran-grafted agarose media for ion exchange chromatography

This work examines the relationship between the physical properties of agarose and dextran-grafted agarose cation exchangers and protein adsorption equilibrium and rates. Four different sulfopropyl (SP) matrices were synthesized using a neutral agarose base material – two based on a short ligand chemistry and two obtained by grafting 10 and 40 kDa dextran polymers. The pore accessibility, determined by inverse size exclusion chromatography (iSEC) with dextran probes, decreases dramatically as a result of the combined effects of crosslinking, dextran grafting, and the introduction of ionic ligands, with pore radii decreasing from 19 nm for the base matrix to 6.1 nm for the 40 kDa dextran-grafted SP-matrix. In spite of this reduction, while the adsorption isotherms were similar, protein uptake rates were greatly increased with the dextran-grafted SP-matrices, compared to SP-matrices based on the short ligand chemistry. The effective pore diffusivities were 4–10 times higher than free solution diffusivity for the dextran-grafted matrices, indicating that the charged dextran grafts result in enhanced protein mass transfer rates.

Mutations in the Tight-Junction Gene Claudin 19 ( CLDN19) Are Associated with Renal Magnesium Wasting, Renal Failure, and Severe Ocular Involvement

Claudins are major components of tight junctions and contribute to the epithelial-barrier function by restricting free diffusion of solutes through the paracellular pathway. We have mapped a new locus for recessive renal magnesium loss on chromosome 1p34.2 and have identified mutations in CLDN19, a member of the claudin multigene family, in patients affected by hypomagnesemia, renal failure, and severe ocular abnormalities. CLDN19 encodes the tight-junction protein claudin-19, and we demonstrate high expression of CLDN19 in renal tubules and the retina. The identified mutations interfere severely with either cell-membrane trafficking or the assembly of the claudin-19 protein. The identification of CLDN19 mutations in patients with chronic renal failure and severe visual impairment supports the fundamental role of claudin-19 for normal renal tubular function and undisturbed organization and development of the retina.

Diffusion in three-dimensionally ordered scaffolds with inverted colloidal crystal geometry

Inverted colloidal crystal geometry has been recently utilized in the design of highly organized 3D cell scaffolds. The regularity of the resulting scaffolds enables computational modeling of scaffold properties. In this work we probe the resistance offered by these scaffolds to nutrient transport, by using Brownian dynamics and Monte Carlo simulations to model the effective nutrient diffusivity. Brownian dynamics simulations indicate that the effective diffusivity for small nutrients in the scaffold, D eff = 0.3 D 0 , where D 0 is the free solution diffusivity. Further, results of Monte Carlo simulations for dilute solutions of larger particles show that the D eff decreases linearly with the size of the particles.

ABC transporters and the blood-brain barrier.

The blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) form a very effective barrier to the free diffusion of many polar solutes into the brain. Many metabolites that are polar have their brain entry facilitated by specific inwardly-directed transport mechanisms. In general the more lipid soluble a molecule or drug is, the more readily it will tend to partition into brain tissue. However, a very significant number of lipid soluble molecules, among them many useful therapeutic drugs have lower brain permeability than would be predicted from a determination of their lipid solubility. These molecules are substrates for the ABC efflux transporters which are present in the BBB and BCSB and the activity of these transporters very efficiently removes the drug from the CNS, thus limiting brain uptake. P-glycoprotein (Pgp) was the first of these ABC transporters to be described, followed by the multidrug resistance-associated proteins (MRP) and more recently breast cancer resistance protein (BCRP). All are expressed in the BBB and BCSFB and combine to reduce the brain penetration of many drugs. This phenomenon of "multidrug resistance" is a major hurdle when it comes to the delivery of therapeutics to the brain, not to mention the problem of cancer chemotherapy in general. Therefore, the development of strategies for bypassing the influence of these ABC transporters and for the design of effective drugs that are not substrates and the development of inhibitors for the ABC transporters becomes a high imperative for the pharmaceutical industry.

Regulated alkali secretion acts in tandem with unstirred layers to regulate mouse gastric surface pH

Background & Aims : The physical basis for the protective pH gradient at the gastric surface is unconfirmed. This study examined the role of mucus, the unstirred layer, and acid/alkali secretion in controlling gastric surface pH in vivo. Methods : Stomachs of anesthetized mice were exteriorized, and exposed gastric mucosa was imaged by confocal microscopy. Results : Accessibility of molecules at the gastric surface was determined by monitoring the decrease in probe fluorescence over time after dyes were removed from perfusate. On dye removal, Cl-NERF (400 molecular weight) fluorescence decreased more slowly at the gastric surface in the presence of mucus (rate constant [k] = 0.08 ± 0.02 per second) than in the absence of mucus (k = 0.15 ± 0.02 per second) or 90 μm distant from the surface (k = 0.22 ± 0.03 per second). In contrast, 70-kilodalton Cl-NERF/dextran washed from the gastric surface more slowly in the presence (k = 0.05 ± 0.01 per second) or absence (k = 0.09 ± 0.01 per second) of mucus compared with 90 μm from the tissue surface (k = 0.36 ± 0.08 per second). Two-photon uncaging of fluorescein near nonsuperfused gastric surface showed that diffusion was not slowed at the gastric surface compared with diffusion in free solution. Surface pH was measured by Cl-NERF ratio imaging. Increasing the superfusion rate decreased the thickness of the surface pH gradient without significantly changing surface pH values, suggesting a pH set point of approximately 4 for control of surface pH. Increasing perfusate pH buffers decreased surface pH toward perfusate values. Conclusions : Proton concentration at the gastric surface is the result of regulating acid/alkali secretion to a set point in combination with an unstirred layer and not by trapping of proton or small-molecular-weight buffers in the unstirred layer.

Modifying the Adsorption Behavior of Polyamidoamine Dendrimers at Silica Surfaces Investigated by Total Internal Reflection Fluorescence Correlation Spectroscopy

Polyamidoamine (PAMAM) dendrimers were modified and tested for use as solution-phase diffusion probes in silica nanostructures. In order for the successful application of dendrimers as solution-phase probes, their interactions with silica surfaces must be understood and controlled, so that the motion of the probe is not influenced by adsorption. Adsorption/desorption kinetics of PAMAM dendrimers and their diffusion in solution near silica surfaces were investigated with total internal reflection fluorescence correlation spectroscopy (TIR-FCS). Dendrimers of generations 3, 5, and 7 were dye-labeled with carboxyrhodamine 6G. Because PAMAM dendrimers are positively charged in solution (having primary amines as end groups), significant adsorption of these molecules to the negatively charged silica surface was observed. Adsorption/desorption rates and the equilibrium constant for adsorption were determined by fitting the autocorrelation functions to a kinetic model. The desorption rate decreases and the absorption equilibrium constant increases with higher dendrimer generation. To reduce the adsorption of these probes to silica surfaces, the labeled dendrimers were reacted with succinic anhydride, converting the primary amine end groups to negatively charged carboxylic acid groups. These carboxylated dendrimers did not detectably adsorb to silica from aqueous solution. TIR-FCS was used to determine their free-solution diffusion constants near silica surfaces, and the corresponding hydrodynamic radii compare favorably with values reported from forced Rayleigh scattering measurements.

Mechanisms of membrane permeabilization by picornavirus 2B viroporin

Cell infection by picornaviruses leads to membrane permeabilization. Recent evidence suggests the involvement of the non-structural protein 2B in this process. We have recently reported the detection of 2B porin-like activity in isolated membrane–protein systems that lack other cell components. According to data derived from these model membranes, four self-aggregated 2B monomers (i.e. tetramers) would be sufficient to permeabilize a single lipid vesicle, allowing the free diffusion of solutes under ca. 1000 Da. Our findings also support a role for lipids in protein oligomerization and subsequent pore opening. The lipid dependence of these processes points to negatively charged cytofacial surfaces as 2B cell membrane targets.

Influence of neutral cyclodextrin concentration on plate numbers in capillary electrophoresis.

A quantitative theory of plate number N in capillary electrophoresis was developed for buffers containing neutral cyclodextrins (CDs) capable of forming inclusion complexes. In the theory, N was modeled by longitudinal diffusion, injection extent, width of the detection window, and the detector time constant. The apparent mobility was modeled as a weighted sum of the mobilities of the free-solution analyte and the inclusion complex. The apparent diffusion coefficient was modeled as a similarly weighted sum. Both the apparent mobility and diffusion coefficient were corrected by functions that compensated for increases of buffer temperature caused by Joule heat. The experimental N's and apparent mobilities of neutral thiourea and of the anions, dansyl D- and L-leucine, dansyl D- and L-aspartic acid, benzoate, and 4-nitrophenolate, were determined in buffers containing from 0 to 15 mM beta-CD. The binding constants, and mobilities and diffusion coefficients of the free-solution analyte and inclusion complex, were calculated as regression coefficients by fitting theory to these determinations. The regression coefficients were shown to have physicochemical meaning, as assessed by literature values, independent measurements, and theoretical predictions. The assessment showed the Nernst-Einstein equation does not relate mobilities and diffusion coefficients at the electrolyte concentration used. The interdependence of mobilities, diffusion coefficients, binding constants, and other dispersion sources was interpreted to evaluate the factors affecting the variation of N with CD concentration. From the interpretation, an approximate equation for N in low-concentration CD buffers was derived. The equation depends on free-solution and inclusion-complex mobilities and diffusion coefficients, the binding constant, the potential difference over the effective capillary length, and the number of plates in a CD-free buffer.

Diffusion of small solutes in the lateral intercellular spaces of MDCK cell epithelium grown on permeable supports.

The diffusion coefficients of four solutes ranging in molecular weight from 238 to 10,000 in the lateral intercellular spaces (LIS) of cultured kidney cells (MDCK) grown on permeable supports were determined from the spread of fluorescence produced after the release of caged compounds by a pulse from a UV laser. Two types of experiments were performed: measurement of the rate of change of fluorescence after releasing a caged fluorophore, and measurement of the change in fluorescence of a relatively static fluorescent dye produced by the diffusion of an uncaged ligand for the dye. Fluorescence intensity was determined by photon-counting the outputs of a multichannel photomultiplier tube. Diffusion coefficients were determined in free solution as well as in the LIS of MDCK cells grown on permeable supports and the hindrance factor, theta, determined from the ratio of the free solution diffusivity to that in the LIS. The hindrance factors for 3000-MW dextran, 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS, MW 524) and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, MW 238) were not significantly different from 1. The diffusion of 10,000-MW dextran was substantially reduced in the LIS with a theta of 5.6 +/- 0.3. Enzymatic digestion by neuraminidase of the sialic acid residues of the glycosylation groups in the LIS increased the diffusivity of the 10,000-MW dextran 1.8-fold indicating hindrance by the glycocalyx. We conclude that small solutes, such as Na(+) and Cl(-), would not be significantly restricted in their diffusion in the LIS and that solute concentration gradients could not develop along the LIS under physiologic conditions.

When can the Ogston-Morris-Rodbard-Chrambach model be applied to gel electrophoresis?

The Ogston-Morris-Rodbard-Chrambach theory of gel electrophoresis is consistent with predictions from the volume averaging method with respect to the equivalence of the accessible volume fraction to the ratios of gel mobility to free solution mobility and the gel diffusion coefficient to free solution diffusion coefficient for the limiting case of small molecule electrophoresis with low electrical fields, low gel concentrations, and nonconductive gel fibers. When these conditions are not valid, more extensive calculations are required to determine the mobility and diffusion coefficient ratios as functions of the geometry and electrical field within the gel. The volume averaging theory shows that it is important to account for the electrical conductivity properties of the fibers that make up a gel electrophoresis medium, and this aspect is consistent with early theories of transport phenomena in gel electrophoresis.

The mitochondrial permeability transition pore and its role in cell death

This article reviews the involvement of the mitochondrial permeability transition pore in necrotic and apoptotic cell death. The pore is formed from a complex of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocase and cyclophilin-D (CyP-D) at contact sites between the mitochondrial outer and inner membranes. In vitro, under pseudopathological conditions of oxidative stress, relatively high Ca2+ and low ATP, the complex flickers into an open-pore state allowing free diffusion of low-Mr solutes across the inner membrane. These conditions correspond to those that unfold during tissue ischaemia and reperfusion, suggesting that pore opening may be an important factor in the pathogenesis of necrotic cell death following ischaemia/reperfusion. Evidence that the pore does open during ischaemia/reperfusion is discussed. There are also strong indications that the VDAC-adenine nucleotide translocase-CyP-D complex can recruit a number of other proteins, including Bax, and that the complex is utilized in some capacity during apoptosis. The apoptotic pathway is amplified by the release of apoptogenic proteins from the mitochondrial intermembrane space, including cytochrome c, apoptosis-inducing factor and some procaspases. Current evidence that the pore complex is involved in outer-membrane rupture and release of these proteins during programmed cell death is reviewed, along with indications that transient pore opening may provoke 'accidental' apoptosis.

Experimental analysis of fluoride diffusion and sorption in clays

Double reservoir diffusion cell tests were employed to estimate parameters for fluoride sorption and diffusion through porous clay media. Tests were conducted on both commercial kaolin and bentonite clays. The bentonite was found to be a better fluoride barrier than kaolin, especially when volumetric changes from swelling were minimised by clamping. Modelled values of effective diffusion in kaolin were typically around 0.015 m 2/yr, which are nearly three times lower than the molecular diffusion in free solution and 10 times greater than the value obtained for clamped bentonite (constant volume and constant confining pressure). Optimised sorption parameters obtained by fitting both linear and non-linear sorption models to diffusion cell breakthrough data were compared with equivalent parameters from batch sorption tests. Both models were equally capable of accurately fitting all experimental data. However, in cases of highly non-linear sorption, care must be taken with the linear model as the sorption parameter depends on the concentration range. Sorption parameters from fluoride batch tests and clay tortuosities from chloride diffusion tests were found to adequately predict fluoride diffusion results. It was found that the modelled parameters for diffusion and partition did not depend significantly on the initial moisture content at compaction of the clay plug. Clay plugs formed through consolidation of a slurry, however, appear to provide more tortuous barriers than compacted clay plugs. Some decline in fluoride retention was observed with increasing ionic strength of solution.