Gel-free Solution Research Articles

Development of stable liquid glucagon formulations for use in artificial pancreas.

A promising approach to treat diabetes is the development of fully automated artificial/bionic pancreas systems that use both insulin and glucagon to maintain euglycemia. A physically and chemically stable liquid formulation of glucagon does not currently exist. Our goal is to develop a glucagon formulation that is stable as a clear and gel-free solution, free of fibrils and that has the requisite long-term shelf life for storage in the supply chain, short-term stability for at least 7 days at 37°C, and pump compatibility for use in a bihormonal pump. We report the development of two distinct families of stable liquid glucagon formulations which utilize surfactant or surfactant-like excipients (LMPC and DDM) to "immobilize" the glucagon in solution potentially through the formation of micelles and prevention of interaction between glucagon molecules. Data are presented that demonstrate long-term physical and chemical stability (~2 years) at 5°C, short-term stability (up to 1 month) under accelerated 37°C testing conditions, pump compatibility for up to 9 days, and adequate glucose responses in dogs and diabetic swine. These stable glucagon formulations show utility and promise for further development in artificial pancreas systems.

Bare Nanocapillary for DNA Separation and Genotyping Analysis in Gel-Free Solutions without Application of External Electric Field

In this work, we demonstrate DNA separation and genotyping analysis in gel-free solutions using a nanocapillary under pressure-driven conditions without application of an external electric field. The nanocapillary is a approximately 50-cm-long and 500-nm-radius bare fused-silica capillary. After a DNA sample is injected, the analytes are eluted out in a chromatographic separation format. The elution order of DNA molecules follows strictly with their sizes, with the longer DNA being eluted out faster than the shorter ones. High resolutions are obtained for both short (a few bases) and long (tens of thousands of base pairs) DNA fragments. Effects of key experimental parameters, such as eluent composition and elution pressure, on separation efficiency and resolution are investigated. We also apply this technique for DNA separations of real-world genotyping samples to demonstrate its feasibility in biological applications. PCR products (without any purification) amplified from Arabidopsis plant genomic DNA crude preparations are directly injected into the nanocapillary, and PCR-amplified DNA fragments are well resolved, allowing for unambiguous identification of samples from heterozygous and homozygous individuals. Since the capillaries used to conduct the separations are uncoated, column lifetime is virtually unlimited. The only material that is consumed in these assays is the eluent, and hence, the operation cost is low.

Evolution of the diffusion field during crystal growth in gel studied by speckle interferometry

Electronic speckle pattern interferometry has been applied for studies of diffusion fields near a single crystal of potassium dihydrogen phosphate (KDP), growing in tetramethoxysilane (TMS) gel. The experimental procedure together with the advantages of speckle interferometry allowed us to study the development of diffusion field after a sudden switch of supersaturation. Comparing experimental data with the results of numerical simulation we have evaluated the linear kinetics coefficient B during gel growth. The obtained value was about three times smaller than that in gel free solution what suggests interaction of crystal surface with the gel net.

Base metals recovery from copper smelter slag by oxidising leaching and solvent extraction

Due to its amorphous structure, smelter slag cannot efficiently be leached with sulphuric acid; the formation of silica gel induces an increase of leach liquor viscosity, difficult pulp filtration and crud formation during solvent extraction. The problem was solved by leaching with sulphuric acid under hydrogen peroxide, which also performs simultaneous iron oxidation and removal. A copper smelter slag from Lubumbashi, Democratic Republic of Congo, containing 1.4% Cu, 0.7% Co, 8.9% Zn and 20.9% Fe(II), has been used in this study. The leaching tests have been carried out at normal pressure on ground slag samples (<100 μm). Silica gel-free solutions containing copper, cobalt and zinc were produced, so that pulp filtration could be easily carried out. The dissolved base metals were successfully extracted from solution by solvent extraction using kerosene Shellsol D70 as diluent. Copper was extracted with LIX 984 and stripped with sulphuric acid solution. Thereafter, cobalt and zinc were collectively extracted with D2EHPA and then separated by selective scrubbing with sulphuric acid solutions of different dilutions. This method provided an overall recovery of 80% Cu, 90% Co and 90% Zn in separated solutions which could be further treated by electrowinning or salt precipitation.

Gel-free experiments of reaction-diffusion front kinetics.

We present a gel-free experimental system to study the kinetics of the reaction front in the A+B-->C reaction-diffusion system with initially-separated reactants. The experimental setup consists of a CCD camera monitoring the kinetics of the front formed in the reaction-diffusion process Cu(2+) + tetra [disodium ethyl bis(5-tetrazolylazo) acetate trihydrate] -->1:1 complex, in aqueous, gel-free solution, taking place inside a 150 microm gap between two flat microscope slides. The experimental results agree with the theoretical predictions for the time dependence of the front's width, height, and location, as well as the global reaction rate.

Spectrophotometric Observations of Gel-Free Reaction Front Kinetics in Confined Geometry

AbstractWe present a new experimental system to study the kinetics of the reaction front in A + B→C reaction-diffusion systems with initially-separated reactants. The set-up is composed of a CCD camera monitoring the kinetics of the front formed in the reaction-diffusion system Cu2++ tetra→1:1 complex (in aqueous, gel-free solution) inside a 150 micron gap between two flat microscope slides. This is basically a two-dimensional system. The results are consistent with the theoretical predictions for the anomalous time dependence of the front's width, height,and location.

Gel growth of lysozyme crystals studied by small angle neutron scattering: case of silica gel, a nucleation inhibitor

Silica gels were used as a media for crystal growth of hen egg white lysozyme crystals. Using small angle neutron scattering, gel-free solutions have been compared to gelled samples in the pre-nucleation period and in the growth period. Characterisation of the protein solution trapped in the gel network shows an adsorption of protein on the gel surface through electrostatic and most probably through hydrogen bonding interactions. The consecutive decrease of concentration is consistent with the decrease of the nucleation rate with respect to gel-free solutions. By modification of the gel surface, we succeeded in increasing the number of nuclei. Growth occurs by incorporation of free molecules and in a second stage, by incorporation of molecules which are progressively desorbed. This provides a relatively constant concentration of the free molecules.

Gel growth of lysozyme crystals studied by small angle neutron scattering: case of agarose gel, a nucleation promotor

In order to understand why hen egg white lysozyme crystals nucleate more easily in agarose gel than in gel-free solutions, we have made a small angle neutron scattering study of gelled and gel-free lysozyme solutions for different supersaturation conditions by varying the temperature, protein and salt (NaCl) concentrations. For higher supersaturations, some kinetics experiments have been done. We have analysed the SANS signal as resulting from two contributions. A first contribution is due to free protein monomers. It shows that concentration and interactions are identical in gel and gel-free media. In this respect, the gel is neutral and supersaturation is not changed. A second contribution is attributed to lysozyme clusters of several tens to several hundreds of nanometers. This contribution increases by increasing supersaturation and, for a given supersaturation, is noticeably stronger in gel than in solution. This effect being not observed when the gel is not set (agarose sol), it has been related to the mechanical properties of the medium. These considerations lead us to propose a link between clustering and nucleation, namely, nucleation would be built out of some of these clusters.

Heterogeneous ethoxylation of cellulose: influence of alkali and available-water concentrations on substituent distributions

O-(2-Hydroxyethyl)cellulose ( 1) as formed by the alkali-catalyzed addition of ethylene oxide to cellulose flock in a slurry process is not uniformly substituted. Most of the ethylene oxide adds to HO-6 in a chain-fashion, so that ∼20% of the d-glucose residues remain totally unsubstituted at 2.0 molar substitution. Consequently, an aqueous solution thickened by 1 is highly susceptible to enzymic degradation. Stepwise decrease in concentration of alkali during etherification gives improved stability to enzymic degradation associated with a more-uniform distribution of hydroxyethyl groups between the three hydroxyl groups of the glucose residues in cellulose. The relative reactivities of hydroxyl groups and patterns of substitution were established by matching the distribution patterns from a stochastic computer-model with the distribution of substituents as determined by chemical analyses [namely hydrolysis with sulfuric acid to determine the percentage of unsubstituted glucose residues (u- 2) and with periodate oxidation for determining the percentage of unsubstituted 2,3-vicinal diol groups per residue]. The reactivities of the three hydroxyl groups at various alkali concentrations in a heterogeneous, slurry-addition process approximate those observed under homogeneous conditions, wherein the reactivities have been determined by tedious chromatographic analyses. In the variable-alkali procedure for addition of ethylene oxide, the amount of water available to the cellulose matrix in the low-alkali ( m) sequence is important both for the stability to enzymic degradation and for obtaining gel-free, thickened, aqueous solutions. Optimal stabilities and gel-free solutions are observed at intermediate water: cellulose ratios of 1.10–1.23. At a ratio of 1.23, the stability to enzymic degradation is less sensitive to percentage variations of u- 2 than in 1 prepared at higher or lower water: cellulose ratios. Although the initial degree of degradation between 1 of high molar substitution prepared at 6.8 m alkali concentration and a similar product prepared under variable alkali conditions may be related to percentage differences of u- 2, the rate and final degree of degradation do not relate to percentage differences of u- 2. An adequate interpretation, utilizing known cellulase turnover-rates, is found in stochastic-model projections of the distribution of consecutive 2 residues not substituted at HO-2. The results indicate that ( 1) more-uniform substitution through equalization of hydroxyl reactivities is achieved by lowering the alkali concentration, and ( 2) more-uniform substitution of 2 of the many cellulose-chains being substituted is achieved by employing an optimal amount of “available” water during etherification.

The isolation and properties of deoxyribonucleoprotein particles containing single nucleic acid molecules

Summary A gel-free solution of deoxyribonucleoprotein was prepared in high yield from calf thymus by maintaining the concentration and ionic strength at very low values and by careful homogenization. The resulting material was examined by a number of different macromolecular methods and found to consist of completely dispersed deoxyribonucleoprotein particles of high asymmetry and containing nearly equal amounts of deoxyribonucleic acid and protein. The molecular weight determined by light scattering was 18·5 million. Upon dissociation the deoxyribonucleic acid obtained had a molecular weight of 8 million suggesting that the deoxyribonucleoprotein particles consisted essentially of one deoxyribonucleic acid molecule and a nearly equal amount of protein. Hydrodynamic investigations and direct electron microscopic examination indicate that the protein is rather evenly distributed along deoxyribonucleic acid chains. Optical rotatory dispersion and infrared spectral studies, while ambiguous, do suggest that a major part of the protein is in the a-helical configuration. It is shown that a large fraction of the histone can be accommodated in sections having the α-helical configuration in the large groove of the double-stranded deoxyribonucleic acid helix and that such a model is consistent with the observations made thus far.