NMR chromatography using microemulsion systems

NMR spectroscopy is an excellent tool for structural analysis of pure compounds. However, for mixtures, it performs poorly because of overlapping signals. Diffusion ordered NMR spectroscopy (DOSY) can be used to separate the spectra of compounds with widely differing molecular weights, but the separation is usually insufficient. NMR "chromatographic" methods have been developed to increase the diffusion separation but these usually introduced solids into the NMR sample that reduce resolution. Using nanostructured dispersed media, such as microemulsions, eliminates the need for suspensions of solids and brings NMR chromatography into the mainstream of NMR analytical techniques. DOSY was used in this study to resolve spectra of mixtures with no increase in line-width as compared to regular solutions. Components of a mixture are differentially dissolved into the separate phases of the microemulsions. Several examples of previously reported microemulsions and those specifically developed for this purpose were used here. These include a fully dilutable microemulsion, a fluorinated microemulsion, and a fully deuterated microemulsion. Log(diffusion) difference enhancements of up to 1.7 orders of magnitude were observed for compounds that have similar diffusion rates in conventional solvents. Examples of commercial pharmaceutical drugs were also analyzed via this new technique, and the spectra of up to six components were resolved from one sample.     

Solubilization of hydrophobic guest molecules in the monoolein discontinuous QL cubic mesophase and its soft nanoparticles

Hydrophobic bioactive guest molecules were solubilized in the discontinuous cubic mesophase (QL) of monoolein. Their effects on the mesophase structure and thermal behavior, and on the formation of soft nanoparticles upon dispersion of the bulk mesophase were studied. Four additives were analyzed. They were classified into two types based on their presumed location within the lipid bilayer and their influence on the phase behavior and structure. Differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS), polarized light microscopy, cryogenic-transmission electron microscopy (cryo-TEM), and dynamic light scattering (DLS) were used for the analysis. We found that carbamazepine and cholesterol (type I molecules) likely localize in the hydrophobic domains, but close to the hydrophobic-hydrophilic region. They induce strong perturbation to the mesophase packing by influencing both the order of the lipid acyl chains and interactions between lipid headgroups. This results in significant reduction of the phase transition enthalpy, and phase separation into lamellar and cubic mesophases above the maximum loading capacity. The inclusion of type I molecules in the mesophase also prevents the formation of soft nanoparticles with long-range internal order upon dispersion. In their presence, only vesicles or sponge-like nanoparticles form. Phytosterols and coenzyme Q10 (type II molecules) present only moderate effects. These molecules reside in the hydrophobic domains, where they cannot alter the lipid curvature or transform the QL mesophase into another phase. Therefore, above maximum loading, excess solubilizate precipitates in crystal forms. Moreover, when type II-loaded QL is dispersed, nanoparticles with long-range order and cubic symmetry (i.e., cubosomes) do form. A model for the growth of the ordered nanoparticles was developed from a series of intermediate structures identified by cryo-TEM. It proposes the development of the internal structure by fusion events between bilayer segments.     

Release of electrolytes from W/O/W double emulsions stabilized by a soluble complex of modified pectin and whey protein isolate

W/O/W double emulsions (DEs) stabilized by charged soluble complexes of whey protein isolate (WPI) and modified pectins were investigated in relation to their stability and the release of two types of electrolytes, NaCl and sodium ascorbate.WPI alone cannot properly stabilize the DEs. The droplet size is relatively large (100 μm) and increases with time. However, addition of modified pectin to form a soluble complex with WPI significantly improved the stability.DEs prepared with two types of oils (medium chain triglycerides (MCT) and R(+)-limonene) were studied by measuring droplet size, creaming, viscosity, and electrolyte release. Irrespective of their very different oil phase nature, both emulsions were stable against coalescence, but R(+)-limonene formed smaller droplets (25 μm) than MCT (35 μm). The electrolyte release rate was significantly higher from the R(+)-limonene that formed DEs with much lower viscosity. R(+)-limonene-DE released 75% of the NaCl after 28 days, while MCT-DE released only 50%. NaCl was released more slowly than sodium ascorbate.Apparently, the release mechanism from R(+)-limonene-DE was found to be “thinning the outer interface and release of the entire inner droplets” while it seems that the release from MCT-DE was slower and “diffusion controlled”.DEs stabilized by WPI/C63 released 12% of the sodium ascorbate after 1 day in milk and remained stable for at least 8 days. However, DEs stabilized with only WPI released about 50% of the sodium ascorbate after 1 day, and phase separated after 8 days.     

High-resolution NMR "chromatography" using a liquids spectrometer

NMR spectroscopy is an excellent tool for the structural analysis of pure compounds. However, for mixtures it performs poorly because of overlapping signals. Diffusion can be used to separate compounds of widely differing molecular weight but the amount of separation is usually insufficient.Addition of a solid medium, analogous to the stationary phase in chromatography, can preferentially slow the diffusion of some components of a mixture permitting separation in the diffusion dimension. However, this would usually require a solid-state NMR spectrometer otherwise the signals would be too broad.Susceptibility matching the solvent to the solid medium yields a spectrum with narrow signals allowing the measurement of a DOSY spectrum with enhanced separation in the diffusion dimension.     

Solution-mediated polymorphic transformation of stearic acid

Polymorphic transformations of stearic acid in their crystallization solutions have been studied. When stearic acid crystals were left in the ethanolic saturated mother liquor, the B-form modification was obtained throughout the whole process. In n-hexane and benzene during the first 2–3 hours, the C-polymorph was the predominant form, later transformed progressively into the B-form, so that towards the end of the experiment the whole precipitate was in the B-form. The proposed explanation is that the transformation is due to dissolution caused by thermal fluctuation in the bath, and subsequent precipitation. The dissolution and reprecipitation was followed up by micro photographs. In the presence of Span 60, only C form was obtained in all the solvents. – As a result of this study it is suggested to pay attention to possible solution-mediated polymorphic transformations of the crystallized material which may cause a misleading interpretation of the factors affecting the crystallization of the fatty acid.     

Newly designed polysiloxane-graft-poly (oxyethylene) copolymeric surfactants: preparation,surface activity and emulsification properties

Two sets of comb-grafted polymeric surfactants based on poly-(methylhydrogen siloxane) (PHMS) and/or poly(dimethylsiloxane) (PHMS-PDMS) were prepared by sililation of the active Si-H group with an active omega-vinyl group of specially designed undecenoic-polyethyleneglycol esters (UPEG) to form newly-designed polysiloxane-grafted-polyethyleneglycol comb-copolymeric surfactants.The hydrophilic moieties are hooked to the hydrophobic backbone through a spacer (undecenoic acid). The variations in the surfactants' structures were in the length and density of the grafted hydrophilic moieties, the chain length (DP) and nature of the hydrophobic backbone.The first 12 different polymeric surfactants (set 1), termed PHMS-UPEG, were found to be ineffective emulsifiers with limited ability to stabilize oil-in-water emulsions. The second set of surfactants, named PHMS-PDMS-UPEG comb-grafted copolymers, significantly reduced the oil-water interfacial tension and effectively stabilized several types of oil-in-water emulsions. The best emulsifier of this set (PHMS-PDMS-52-UPEG-45), seems to be the one whose anchor backbone (PHMS-PDMS) dissolves (rather than spreads) in the oil phase, and whose stabilizing moieties are sufficiently long (45 EO units) and hooked to the silicone backbone at high density (52% substitution).     

Sugar-Ester Nonionic Microemulsion: Structural Characterization

Surfactants containing sugar components and fatty acids satisfy the quality standards for food application. The food grade sugar ester in this study is a commercial sucrose monoester of stearic acid (abbreviated SES), the oil phase consists of a 1:1 mixture of n-tetradecane and l-butanol. The originally planned food grade oil, a medium chain triglyceride, is substituted by tetradecane because tetradecane is available as a fully deuterated product, which is necessary for some structural investigations. The investigated system is solid at room temperature, but liquefies and structures into a homogeneous microemulsion when heated to above 37 degrees C. The structural characterization of such microemulsions is the aim of this work. The established methods for this purpose are scattering methods, such as small-angle scattering of X-rays and neutrons and dynamic light scattering. These scattering techniques can be used to obtain valuable information on the size, shape, and internal structure of colloids and complex fluids. We started our investigation with the pseudobinary system SES, tetradecane and l-butanol, varying the SES content. The scattering results show that the sugar ester form inverse globular micelles in the oil phase. The size of these micelles is about 6 nm. While the size is nearly constant in a wide SES concentration regime (5 up to 40% surfactant), the volume or aggregation number increases significantly with SES. This is explained by an increasing replacement of l-butanol molecules by sugar-ester molecules in the micelles formed. Moreover, it can be shown that these micelles strongly overlap. Their center-to-center distance is about 3.8 nm at 40% SES at a micellar diameter of 6 nm. The micellar overlap leads to a highly reduced diffusion of the micelles as was found with dynamic light scattering. When incorporating water in the micellar core, the micelles swell up to about 10 nm and the shape of the aggregates becomes more and more elongated with higher water content. Copyright 2001 Academic Press.     

Low viscosity reversed hexagonal mesophases induced by hydrophilic additives

This study reports on the formation of a low viscosity H(II) mesophase at room temperature upon addition of Transcutol (diethylene glycol mono ethyl ether) or ethanol to the ternary mixture of GMO (glycerol monooleate)/TAG (tricaprylin)/water. The microstructure and bulk properties were characterized in comparison with those of the low viscosity HII mesophase formed in the ternary GMO/TAG/water mixture at elevated temperatures (35-40 degrees C). We characterized the role of Transcutol or ethanol as inducers of disorder and surfactant mobility. The techniques used were rheology, differential scanning calorimetry (DSC), wide- and small-angle X-ray scattering (WAXS and SAXS, respectively), NMR (self-diffusion and (2)H NMR), and Fourier transform infrared (FTIR) spectroscopies. The incorporation of either Transcutol or ethanol induced the formation of less ordered HII mesophases with smaller domain sizes and lattice parameters at room temperature (up to 30 degrees C), similar to those found for the GMO/TAG/water mixture at more elevated temperatures (35-40 degrees C). On the basis of our measurements, we suggest that Transcutol or ethanol causes dehydration of the GMO headgroups and enhances the mobility of the GMO chains. As a result, these two small molecules, which compete for water with the GMO polar headgroups, may increase the curvature of the cylindrical micelles and also perhaps reduce their length. This results in the formation of fluid H(II) structures at room temperature (up to 30 degrees C). It is possible that these phases are a prelude to the H(II)-L(2) transformation, which takes place above 35 degrees C.     

The effect of structural variation of alcohols on water solubilization in nonionic microemulsions 2. Branched alcohols as solubilization modifiers: results and interpretation

In this second part of a paper dealing with the effect of branched alcohols on solubilization, an attempt has been made to provide explanations of experimental data related mostly to the system Brij 97/branched alcohol + dodecane = 1:1 (by weight)/water at 27+/-0.2 degrees C. Applying the Hou-Shah mechanism it was shown that for many C4-C6 branched alcohol isomers having one methyl branch, solubilization behavior is readily interpreted by assuming control of the critical radius, R(c). Two parameters, both included in the definition of the branching factor, F(b) (which was treated in the first part of the paper), were also used to analyze solubilization data. The first, l(i), is defined as the distance from the free end of the alcohol molecule to the methyl branch. The second, d, is virtually N(A), the chain length of the alcohol. When l(i)>3, the solubilization becomes dominated by the natural radius of curvature, R0. Also, we have suggested that for R(c)-control, solubilization will be enhanced in direct proportion to the distance d-l(i), whereas for R0-control, solubilization will increase with decreasing d-l(i). The validity of our assumptions was demonstrated in many cases. Some examples of the more complicated case of double branching (two methyl groups along the alcohol chain) were also analyzed.     

Templated cocrystallization of cholesterol and phytosterols from microemulsions

A major cause of cardiovascular disease is high cholesterol (CH) levels in the blood, a potential solution to which is the intake of phytosterols (PS) known as CH-reducing agents. One mechanism proposed for PS activity is the mutual cocrystallization of CH and PS from dietary mixed micelles (DMM), a process that removes excess CH from the transporting micelles. In this study, microemulsions (MEs) were used both as a model system for cocrystallization mimicking DMM and as a possible alternative pathway, based on the competitive solubilization of CH and PS, to reduce solubilized CH transport levels from the ME. The effects of different CH/PS ratios, aqueous dilution, and lecithin-based MEs on sterol crystallization were studied. The precipitated crystals from the ME-loaded system with PS alone and from that loaded with 1:1 or 1:3 CH/PS mixtures were significantly influenced by ME microstructure and by dilution with aqueous phase (X-ray powder diffraction (XRD) and differential scanning calorimetry (DSC) results). No new polymorphic structures were detected apart from the corresponding sterol hydrates. Mixed crystal morphology and the habit of the precipitated sterols were strongly affected by the CH/PS ratio and the structures of the diluted ME. As the amount of PS in the mixture increased or as the ME aqueous dilution proceeded, precipitated crystal shape became more needle-like. The mixed sterols seemed to be forming eutectic solids.