To determine the reason why the adsorption of ethylene glycol on organo-smectites does not result in an expansion along the c-axis of the clays, smectites containing relatively small organo-ammonium ions (lauryl-, benzyl-, dibenzyl-, and dicyclohexylammonium), larger organic cations (dimethylbenzyloctadeyl- and methylbenzyldioctadecylammonium), and the heterocyclic organo-ammonium ion 1,4a-dimethyl-7-isopropyl-1,2,3,4,4a,9,10,10a-octahydro-1-phenanthrenemethylammonium and the corresponding ethoxylated compound were exposed to ethylene glycol vapor for up to several months and examined by X-ray powder diffraction (XRD), surface area, and thermogravimetric methods. Weight loss data showed that all samples adsorbed ethylene glycol. XRD data for oriented samples indicated that lauryl-, benzyl-, dicyclohexyl-, and ethoxylated heterocyclic ammonium clays expanded by one layer of ethylene glycol and that methylbenzyldioctadecylammonium smectite expanded by two layers. Dibenzyl-, dimethylbenzyloctadecyl-, and heterocyclic smectites did not expand because the clay oriented in such a manner as to leave free clay surface between the organo-ammonium cations.

The thickness of the two-layer ethylene glycol complex of dioctahedral smectites varies under room conditions between 17.3 and 16.5 Å because of such factors as layer charge density, type of interlayer cation, and relative humidity. Neglecting this variability can give up to 30% error in the X-ray powder diffraction estimation of the smectite:illite ratio of the mixed-layer structures. Three methods have been developed for the interpretation of X-ray powder diffraction patterns of glycolated mixed-layer illite/smectite which take layer-spacing variability into account. The methods include a technique for quantifying the degree of layer ordering. In addition, the proposed techniques minimize the error due to the influence of domain size on positions of reflections. The experimental error can be kept below 5% or below 1% smectite layers, depending on the method applied, provided that the peak positions are measured with the accuracy of ± 0.02°2θ.

The structure of sepiolite from a piston core obtained on Ninetyeast Ridge in the Indian Ocean was modified by exposure to ethylene glycol vapor. With ethylene glycol, the sepiolite Oil X-ray powder diffraction peak expanded from 12.4 to 12.8 Å, and the 130 peak contracted from 4.53 to about 4.45 Å. The cell modification is consistent with concomitant expansion along the c-axis and contraction along the b-axis. This structural distortion is not permanent, however, inasmuch as the sepiolite returned to its original state 6 to 12 hr after it was removed from the ethylene glycol-saturated atmosphere.

X-ray powder diffraction patterns for many interstratified kaolinite/glycolated smectites were calculated by changing combinations of probabilities and transition probabilities of two component layers. Reichweite = 0 and Reichweite = 1 structures were investigated. The calculated d-values were plotted, with PKS (the probability that a smectite layer succeeds a kaolin layer given that the first layer is a kaolinite layer) and PSK as the axes of coordinates. These d-values were then linked into equal d-value curves on a graph. Four equal d-value diagrams for mixed reflections ranging from 34.0 to 17.1 Å, from 8.5 to 7.2 Å, from 6.13 to 5.68 Å, and from 3.540 to 3.401 Å were constructed. Two examples of identifying natural kaolinite/smectite minerals using these diagrams are presented.

X-ray powder diffraction (XRD) patterns for many interstratified mica/glycolated smectites were calculated by changing combinations of probabilities and transition probabilities of two-component layers. Three basal XRD reflections, 5. l°–7.6°2θ (p1), 8.9°–10.2°2θ (p2), and 16.1°–17.2°2θ (p3) were selected for the quantification curves. A distinct relationship exists between Δ2θ, (p2–p1) and Δ2θ2 (p3–p2) which shows systematic changes with expandability at constant Reichweite values. The calculated values were plotted with Δ2θ1 and Δ2θ2 as the axes of coordinates, and quantification curves were calculated. The components and stacking parameters of mica/smectites were estimated easily using this diagram. Probabilities of existence of component layers and their transition probabilities for Reichweite (R=0) and (R=l) structures, and special cases of R=2 and R=3 structures were obtained.

The effects of relative humidity (RH) on the expansion of Mg-smectite, equilibrated with low vapor pressure of ethylene glycol (EG), was studied. Four smectite samples were equilibrated with EG vapor from: (1) saturated vapor of pure EG, (2) an EG-CaCl2 solvate, and (3) 0.05 relative EG vapor pressure at 65°C. X-ray powder diffraction (XRD) analyses of the clays under different RH conditions indicated that without EG, the basal spacing of Mg-smectite samples could only be expanded to 16 Å, even at 0.97 RH. The basal spacing of Mg-smectite samples equilibrated with saturated EG vapor was expanded to 17.1 Å and was not significantly affected by RH in the range 0.1–0.9. The basal spacing of the Mg-smectite samples equilibrated with vapor from the EG-CaCl2 solvate and 0.05 relative EG pressure at 65°C was expanded by an amount that depended on the RH during the XRD analysis. The basal spacing increased from 14.2 to 17.1 Å as the RH increased from 0.6 to 0.75, except for sample API 23, which expanded to 17.1 Å at RH > 0.9. This sample did not expand beyond 16 Å when it was equilibrated with 0.05 EG relative vapor pressure. Sorption of moisture from air caused the one-layer EG-Mg-smectite complex (basal spacing = 14.1 Å) to rearrange itself to a double-layer EG-Mg-smectite complex (basal spacing = 17.1 Å). A small amount of the adsorbed EG in Mg-smectite, much less than was needed to cover a one-layer of the interlayer surfaces, caused an expansion of the basal spacing to 17.1 Å at high RH during the XRD analysis. The minimum amount of adsorbed EG which caused the one-/two-layer EG complex conversion was about 20–30 mg EG/g. The conversion was fast (<5 min) and was relatively reversible.

X-ray powder diffraction intensities for many interstratified structures of mica/smectite and chlorite/smectite were calculated by changing combinations of probabilities and transition probabilities of two component layers, respectively. The calculated d-values were plotted with PMS and PSM as the axes of coordinates for mica/smectites (where M is a mica layer and S is a smectite layer). These d-values were then linked into equal d-value curves on a graph. Three equal d-value diagrams ranging from 32.5 to 24.5 Å, from 15.4 to 10.25 Å, and from 3.365 to 3.08 Å were constructed for mica/smectites. Several diagrams were also constructed for mica/glycolated-smectites and chlorite/smectites using the same techniques. PMS and PSM values of mica/smectite producing 26.8- and 12.6-Å reflections in its X-ray powder diffraction pattern were obtained from the coordinates of the intersection of the 26.8-Å line of the first diagram and the 12.6-Å line of the second diagram. The components and stacking parameters of mica/smectites and chlorite/smectites were estimated easily using these diagrams. Interstratified mica/smectites were quantified in the air-dry and glycolated states, and chlorite/smectites in the glycolated state. Stacking parameters obtained by this method agreed well with those obtained by MacEwan's method. Stacking parameters for Reichweite (R=0) and (R= 1) structures were obtained.

A stable 8.4 Å hydrate of kaolinite was prepared by exchanging ethylene glycol for water in the 10.8 Å intercalate of ethylene glycol in kaolinite. The hydrate of kaolinite was characterized by XRD, FTIR and TGA/DSC. From the TGA data, one can estimate that there is 0.60 water molecule per Al2Si2O5(OH)4 units. The IR data suggest a similarity of the local environment of the intercalated water in this 8.4 Å hydrate of kaolinite and the 8.4 Å hydrate of nacrite previously described by Wada (1965).

Expansion properties often homoionic smectites that differed in amount and location of layer charge were examined by X-ray powder diffraction analysis at various relative humidities, or after glycerol or ethylene glycol solvations. Except for K-samples with glycerol solvation, and Na- and Ca-samples with ethylene glycol, differences in the basal spacings are observed in samples having similar layer charge. These results show that the basal spacings are larger when the layer charge is located in octahedral sites than when it is in tetrahedral sites. This suggests that expansion is due to the combined effects of the charge location and amount.

The effects of layer charge magnitude and location on expansion were represented by an energy change (expansion energy: ΔEr) during the hydration and solvation processes. Plots of basal spacings versus ΔEr show a reasonable relationship; the spacings generally decrease stepwise as the value of ΔEr increases. The basal spacings of K-samples with glycerol solvation, Na-saturated and K-saturated samples at 100% RH are apt to contract stepwise with increasing value of ΔEr. For these samples, the figures showing the relationship between each expanded phase and the charge characteristics are obtained from the isoquants of ΔEr, given the boundary of the expanded phases. A behavior test using these figures may be combined with the Greene-Kelly test to estimate the amount and the location of the layer charge of common smectites.

A new, well-ordered, thermally robust ethylene glycol intercalate of kaolinite was formed by refluxing the dimethyl sulfoxide intercalate of kaolinite (Kao-DMSO) with dry ethylene glycol (EG). This new phase (Kao-EG 9.4 Å) which is characterized by a d001 of 9.4 Å is distinct from a previously reported ethylene glycol intercalated phase of kaolinite (Kao-EG 10.8 Å) which has a d001 of 10.8 Å. The characterization of these two phases was studied by XRD, NMR, FTIR, and TGA/DSC. It was found that the concentration of water in the ethylene glycol reaction media played a crucial role in governing which of the phases predominated. Water favored Kao-EG 10.8 Å formation, while anhydrous conditions favored the formation of Kao-EG 9.4 Å. It is hypothesized that Kao-EG 9.4 Å is a grafted phase resulting from the product of the condensation reaction between an aluminol group on the interlamenar surface of kaolinite and the alcohol group of ethylene glycol. Ethylene glycol units would be attached to the interlamellar surface of kaolinite via Al-O-C bonds. The Kao-EG 9.4 Å phase was found to be resistant to both thermal decomposition up to 330°C and also, once formed, in the absence of interlamellar water molecules, to decomposition by hydrolysis in refluxing water.

Organo-clays represent a special challenge for molecular simulations because they require accurate representation of the clay and the organic/aqueous sections of the model system and accurate representation of the interactions between them. Due to the broad range of force-field models available, an important question to ask is which sets of parameters will best suit the molecular modeling of the organo-intercalated smectites? To answer this question, the structure of the ethylene glycol (EG)-smectite complex is used here as a testing model because the intercalation of EG in smectites provides a stable interlayer complex with relatively constant basal spacing.

Three smectite samples with substantially different layer charge and charge localization were selected for X-ray diffraction (XRD) measurements. Their molecular models were built and molecular-dynamics simulations performed using various combinations of the organic force fields (CGenFF, GAFF, CVFF, and OPLS-aa) with ClayFF and INTERFACE force fields used to describe smectites. The simulations covered a range of different EG and water contents. For every structure, the density distribution of interlayer species along the direction perpendicular to the layer plane was calculated and then used to optimize the XRD patterns for these simulated models.

A comparison of these results with experimental XRD patterns shows very large discrepancies in the structures and basal spacings obtained for different layer charges as well as for different force fields and their combinations. The most significant factor affecting the accuracy of the calculated XRD patterns was the selection of the clay-mineral force-field parameters. The second important conclusion is that a slight modification of the basal oxygen parameters for non-electrostatic interactions (increase of their effective atomic diameters) may be a simple and straightforward way to improve significantly the agreement between the modeled XRD patterns with experiments, especially for high-charge smectites. Generally, among organic force fields, the least accurate results were obtained with CGenFF. For unmodified ClayFF, its combination with GAFF gave the best results, while the two other sets (OPLS-aa and CVFF) gave the best results in combination with ClayFFmod. The INTERFACE and INTERFACEmod produced much better results for low-charge montmorillonite than for high-charge smectites.

X-ray powder diffraction patterns of halloysite (7 Å) are characteristically altered following solvation with ethylene glycol. Some effect was first noted in the classic work of MacEwan but its value in the unequivocal identification of halloysite (7 Å ) seems to have been overlooked subsequently. The response to ethylene glycol solvation involves a decrease in the intensity (peak height) of the peak at ∼7.2 Å and an increase in the intensity (peak height) of the peak at ∼3.58 Å thus narrowing the 7.2 Å /3.58 Å peak height intensity ratio. For pure samples of halloysite, this ratio is narrowed by an average of ∼50%. This distinctive change is related to the interstratified nature of halloysite (7 Å), specifically the presence of ‘residual’ interlayer water, i.e. halloysite (10 Å), which can be replaced with ethylene glycol so forming 10.9 Å layers, a spacing that is almost exactly one and a half times the thickness of dehydrated (7.2 Å) layers which do not imbibe ethylene glycol. Thus the separation between the 001 peaks in the 7.2 Å /10.9 Å interstratification is increased and the 0027.2 (3.58 Å) and 00310.9 (3.63 Å) peaks become more or less coincident, compared to the 7.2 Å /10 Å interstratification, i.e. the partially hydrated state. The widespread use of ethylene glycol solvation in clay mineral studies makes it a particularly useful and simple test to determine the presence of halloysite. Pure halloysites should be readily identifiable and experiments indicate a ‘routine’ sensitivity of ∼20% halloysite in mixtures with kaolinite, although this will depend on factors such as ‘crystallinity’ and could be improved with careful attention to intensity measurements. It is proposed to call this phenomenon the ‘MacEwan effect’ in honour of its discoverer Douglas Maclean Clark MacEwan.

While vitrification has become the method of choice for preservation of human oocytes and embryos, cryopreservation of complex tissues and of large yolk-containing cells, remains largely unsuccessful. One critical step in such instances is appropriate permeation while avoiding potentially toxic concentrations of cryoprotectants. Permeation of water and small non-charged solutes, such as those used as cryoprotectants, occurs largely through membrane channel proteins termed aquaporins (AQPs). Substitution of a Thr by an Ala residue in the pore-forming motif of the zebrafish (Dario rerio) Aqp3b paralog resulted in a mutant (DrAqp3b-T85A) that when expressed in Xenopus or porcine oocytes increased their permeability to ethylene glycol at pH 7.5 and 8.5. The main objective of this study was to test whether ectopic expression of DrAqp3b-T85A also conferred higher resistance to cryoinjury. For this, DrAqp3b-T85A + eGFP (reporter) cRNA, or eGFP cRNA alone, was microinjected into in vivo fertilized 1-cell mouse zygotes. Following culture to the 2-cell stage, appropriate membrane expression of DrAqp3b-T85A was confirmed by immunofluorescence microscopy using a primary specific antibody directed against the C-terminus of DrAqp3b. Microinjected 2-cell embryos were then cryopreserved using a fast-freezing rate and low concentration (1.5 M) of ethylene glycol in order to highlight any benefits from DrAqp3b-T85A expression. Notably, post-thaw survival rates were higher (P<0.05) for T85A–eGFP-injected than for -uninjected or eGFP-injected embryos (73±7.3 vs. 28±7.3 or 14±6.7, respectively). We propose that ectopic expression of mutant AQPs may provide an avenue to improve cryopreservation results of large cells and tissues in which current vitrification protocols yield low survival.

The process of cooling and cryopreservation of prawn embryos is a viable alternative for a continuous supply of larvae for freshwater prawn farming ponds. However, studies involving the application of those techniques as well as on toxicity of cryoprotectants in freshwater prawn embryos are scarce. Thus, this study aims to test the toxicity of methylic alcohol (MET), dimethyl sulfoxide (DMSO) and ethylene glycol (EG) on Macrobrachium amazonicum embryos. For the present experiment, pools of embryos were taken from 15 M. amazonicum females and were divided into three groups and tested in duplicate at concentrations of 10, 5, 3; 1, 0.5 or 0.1%. Toxicity tests were conducted for 24 h in Falcon® pipes to obtain the lethal concentration for 50% of the larvae (LC50). After the set period for testing, random samples of embryos were removed for morphological analysis under stereoscopic microscopes. Results were analysed using analysis of variance (ANOVA) and Tukey's test at a 5% significance level and Trimmed Spearman-Karber Analysis to determine LC50-24 h. DMSO toxicity tests revealed that 5% and 10% concentrations showed the highest toxicity and differed from the control (P ≤ 0.05), 24h-LC50 was 437.4 ± 14.4 µL. MET was less toxic among the tested cryoprotectants and concentrations did not allow the determination of its LC50-24h. For tests with EG, concentrations of 3, 5 or 10% solutions resulted in a 100% mortality to tested embryos; EG was the tested cryoprotectant with the highest toxicity, with an LC50-24h average of 81.91 ± 35.3 µl.