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Stripping interfering sugars from samples using adapted bacteria.

Bacteria adapted to individual sugars quickly remove targeted sugars--stripping them--from samples in which unwanted sugars interfere. Adapted bacteria are equivalent to specific reagents for removal of sugars down to bacterial Km values, micromolar to submicromolar concentrations. Bacterial stripping is a simple method, useful when background sugars in micro-to millimolar concentrations (or larger) interfere with analysis of sought-for sugars. Bacteria such as Escherichia coli and Klebsiella are easily adapted to individual sugars such as lactose, fructose, etc., by growing the bacteria on them. Hence one can easily create (and store) many kinds of cells ready to sponge up or strip out unwanted compounds. E. coli specifically remove several sugars from samples containing 100-500 nmol of sugars, using 1-5 mg of adapted cells, and 25 degrees C temperatures. Stripping requires 1-5 min and consists of mixing cells and sample, spinning down the cells, and withdrawal of stripped supernate. A 1-5 min interval is adequate for uptake and stripping, but far too short for cells to metabolize the sugars that were taken up. Hence the cells do not leak metabolites, but act as specific adsorbants without injection of appreciable byproducts into the sample.

Adaptation, Biological↗

Interaction between viruses and monoclonal antibodies studied by surface plasmon resonance.

An automated biosensor system designed for measuring molecular interactions in real time and without any labelling of the reactants has been used to study the interaction of two animal viruses (vaccinia virus and poliovirus) and two plant viruses (cowpea mosaic virus and tobacco mosaic virus) with monoclonal antibodies. Using monoclonal antibodies specific for different conformational states of viral protein, it was found that the virus particles retained their conformational integrity when immobilized on the dextran matrix present on the sensor chip. Compared to conventional solid phase immunoassays, in which immobilized proteins are usually partly denatured, the biosensor system presents several advantages for studying virus-antibody interaction.

Antibodies, Monoclonal↗

The estimation of virus density in isopycnic cesium chloride gradients.

Methods for estimating the density of virus particles after isopycnic centrifugation in cesium chloride gradients were examined. If solutions were prepared in different buffers, the standard tables relating refractive index and density, based on solutions prepared in water, did not give consistent estimates even if corrections were made for the greater refractive indices of the buffers. Depending on the method and buffer used, estimates of the density of cricket paralysis virus, an insect picornavirus, differed by as much as 0.05 g/ml. However, consistent estimates were obtained if separate regression equations were derived to relate refractive index and density for each different buffer solution of cesium chloride. This has wider implications and could account for density differences or variations in estimates reported for many other viruses.

Centrifugation, Isopycnic↗

Hybrid diffractive-refractive achromatic spectacle lenses.

The possible utility of high-power hybrid achromatic spectacle lenses, each consisting of a contact combination of refractive and diffractive components, is considered. It is shown that such a combination can, in principle, minimize blur due to transverse chromatic aberration in high-power prescriptions such as those for aphakes and high myopes. The combination has essentially the same thickness and weight as a conventional singlet lens. Some practical problems relating to hybrid designs are discussed.

Equipment Design↗

Analysis of optical transmission by 400-500 nm visible light into aesthetic dental biomaterials.

The penetration of visible light into dental biomaterials is an essential factor in photoinitiation of setting reactions and in the optical aspects of dental aesthetics. Light of visible blue wavelengths, 400-500 nm, has been applied at normal angles to 0.2-5.0 mm sections of human dentine and representative ceramic, polymerceramic composites and hybrid glass-polyalkenoate materials. The integrated optical transmission has been determined for each material section. The data have been converted to absorbance values and analysed to check for mathematical conformity to the Beer-Lambert Law. It is found that conformity (typically, P < 0.01) to the linear Beer-Lambert Law is only attained by making a substantial correction for the intensity of light reflected from the surface of aesthetic biomaterials. This is otherwise expressed by distinguishing between true and apparent absorbance. From linear regression of apparent absorbance with section thickness, the intercept depends upon the logarithm of the surface-reflection ratio. This factor ranges from 30% to 90% in the materials investigated. It follows that there is a high degree of inefficiency in the transmission of visible light into and through aesthetic biomaterials for the purposes of photoactivation using existing technology. Means by which this limitation and inefficiency may be reduced are discussed. While the reflectivity of aesthetic biomaterials has been perceived by dental practitioners, the magnitude of this effect and its implications in connection with light-cured materials have not been analysed and emphasized hitherto.

Absorption↗

Molecular characterisation of kappa- and lambda-carrageenan by gel permeation chromatography, light scattering, sedimentation analysis and osmometry.

Gel permeation chromatography, in conjunction with a double detection system involving a low angle laser light scattering apparatus (LALLS) and a refractive index monitoring device (RI), has been used to obtain both the molecular weight and the molecular weight distribution of sodium salts of kappa-carrageenan and lambda-carrageenan in saline solutions. The results, Mw and Mn, are in excellent agreement with independent determinations of molar mass based on static light scattering experiments, sedimentation-diffusion analysis and osmometry. The relevance of the data is discussed with respect to current problems in carrageenan research.

Carrageenan↗

Fluorescence decay of DPH in lipid membranes: influence of the external refractive index.

The radiative decay rate of a fluorescent probe in an optically thin layer is known to depend on the orientation of the probe and on the refractive indices inside and outside the layer (W. Lukosz, Phys. Rev. B 22 (1980) 3030). Fluorescent probes in phospholipid bilayer membranes approximate such a system. The natural lifetime is expected to vary with the refractive index of the medium surrounding the bilayer. The lifetime variation with the refractive index depends on the orientation of the fluorescent probe. This can be used to retrieve the second-rank orientational order parameter, . The fluorescence decay of all-trans 1,6-diphenyl-1,3,5-hexatriene in L-alpha-dipalmitoyl-phosphatidylcholine large unilamellar vesicles (LUVs) was measured at a temperature well below that of the phase transition. The refractive index of the medium was varied by addition of glycerol or sucrose. The observed change of decay time with the refractive index followed the theoretical prediction. The value of the order parameter, , recovered is significantly lower than that obtained from fluorescence polarization data. Possible reasons for this disagreement are discussed.

1,2-Dipalmitoylphosphatidylcholine↗

High fat diets and mouse colon mucosal membranes: a centrifugation study.

Female Swiss mice were fed on a control diet (laboratory chow, 6% fat) or high fat diets (laboratory chow with added fat, principally corn oil, to produce a fat content of 16% or 23%). Homogenate of colon mucosa was fractionated by isopycnic banding on a linear sucrose gradient and the distribution of 5'-nucleotidase determined. Two main peaks were detected which we interpret as belonging to basolateral plasma membrane (BLPM) and brush borders. A high fat diet did not affect the relative sizes of the peaks or the median density of the BLPM peak but did significantly reduce the density of the brush border peak from 1.208 g/ml to 1.197 g/ml.

5'-Nucleotidase↗

Growth related changes to functional parameters in the bovine lens.

Dimensions, volumes and protein contents were measured for bovine lenses with wet weights ranging from 0.17-3.07 g (2 months gestation to 19 years post-natal). All increase in a non-linear fashion. The lens becomes flatter with age due to a more rapid increase in the equatorial plane, but the ratio of anterior to posterior sagittal distances remains constant (1.19). The radius of curvature increases from 4.9 to 15 for the anterior surface and from 4.4 to 13 for the posterior. Protein content increases more rapidly than volume resulting in an increased average protein concentration from around 18% in the early prenatal lens to nearly 50% in the 19 year old. Total protein content (TPC) was found to be related to wet weight (We) according to the equation, TPC = 0.3We1.33. It is suggested that TPC is a better parameter for describing growth than wet weight or age. The refractive index, in the equatorial plane, increases towards the centre, from 1.38 at the edge of the lens. The maximum index, in the centre, increases with lens size up to 1.474 in the largest lens studied. This corresponds to a protein concentration of 70%. In all lenses, refractive index and protein concentration gradients were superimposable when plotted from the outside towards the centre. The optical performance of the lenses was assessed by measuring the back focal length which increases gradually from 24 to 51.5 mm over the 0.17 to 3.07 g size range. This was attributed to the increased radii of curvature.

Aging↗

High-performance liquid chromatographic determination of metabolic products for fermentation control of mammalian cell culture: analysis of carbohydrates, organic acids and orthophosphate using refractive index and ultraviolet detectors.

A method for the determination of carbohydrate substrates and excreted metabolic end-products of cell culture supernatants using a strong cation-exchange column in the H+ form has been developed. Organic acids and carbohydrates can be determined in addition to orthophosphoric acid. Pyrrolidone carboxylic acid, resulting from chemical conversion of the amino acid glutamine during the incubation of fresh medium and during the fermentation process, can be determined. The chromatographic method allows the correction of glutamine uptake values for physiological studies. Measured values of pyrrolidone carboxylic acid in supernatants of human hybridoma cell line show that it cannot be consumed by the cells. This technique allows the separation of major metabolites used in process optimization. Peak homogeneity is proved by on-line monitoring of the effluent with an ultraviolet (214 nm) and a refractive index detector connected in series.

Acids↗

Simple cation-exchange high-performance liquid chromatography optimized to the measurement of metabolites in the effluents from perfused rat livers using refractive index and ultraviolet detectors.

A high-performance liquid chromatographic method designed to analyse effluents from perfused organs is described. In the case of rat liver, compounds released by the liver are readily separated and quantitated, using a strong cation exchanger (Aminex HPX 87H), two detectors connected in series (ultraviolet detector at 210 nm and refractive index detector), and by optimizing the concentration of sulphuric acid in the mobile phase. Chromatographic conditions described in the present work enable the quantitation, in a single run, of metabolites derived from the tricarboxylic acid cycle, glycolysis, ketogenesis, adenine nucleotides catabolism and ethanol oxidation. The advantage of this method stems from its ease of implementation, sensitivity and flexibility.

Adenine↗

Determination of neutral oligosaccharide fractions from human milk by gel permeation chromatography.

A gel permeation chromatographic method for quantifying neutral oligosaccharide fractions from human milk has been developed. Oligosaccharides from monofucosyllactoses to trifucosyllacto-N-hexaoses were separated according to size on a Fractogel TSK HW 40 (S) column. Refractive index detection of monofucosyllactoses to difucosyllacto-N-tetraoses yielded a constant mass response factor of ca. 1 relative to glucose. After the addition of glucose as an internal standard, oligosaccharides were isolated from human milk by ethanol precipitation or two ultrafiltration procedures. The oligosaccharide concentrations found by the ultrafiltration procedures were significantly lower (significance level 0.05) than those determined by the ethanol precipitation procedure.

Carbohydrate Sequence↗

Comparison of the carbohydrate biological response modifiers Krestin, schizophyllan and glucan phosphate by aqueous size exclusion chromatography with in-line argon-ion multi-angle laser light scattering photometry and differential viscometry detectors.

A major barrier to the development, preclinical and clinical application of natural carbohydrate biological response modifiers has been the difficulty involved in accurately characterizing carbohydrate polymers with molecular masses ranging from 10(4) to 10(7) g/mol. Herein, we employed size exclusion chromatography with multi-angle laser light scattering and differential viscometry to compare and contrast structural properties of the biological response modifiers Krestin, schizophyllan and glucan phosphate. Krestin, schizophyllan and glucan phosphate exhibit significant differences in molecular mass moments, molecular mass distribution, polymer sizes, intrinsic viscosity and perhaps their solution behaviour. This knowledge of precise physicochemical data is required for a better understanding of the properties and higher structure of complex carbohydrate biological response modifiers.

Chromatography, Gel↗

The chemistry of iomeprol and physico-chemical properties of its aqueous solutions and pharmaceutical formulations.

The synthesis and the chemical characterization of iomeprol, a new triiodinated nonionic radiographic contrast agent, are reported. The physico-chemical properties both of aqueous solutions of the pure compound and of its pharmaceutical formulations are presented and these last data are compared with those of other contrast media. The pharmaceutical development of the product is described. Iomeprol is obtained via a synthesis which is particularly friendly to the environment. The compound shows an unusually high solubility which allows the formulation of contrast media with the lowest osmolalities and viscosities as compared with corresponding contrast media of the same category. In view of these favourable characteristics and of the remarkably high stability of its solutions, iomeprol for injection is formulated also at 400 mgI/ml, the highest concentration so far available on the market for non-ionic contrast media.

Anilides↗

HPLC study of the impurities present in different ursodeoxycholic acid preparations: comparative evaluation of four detectors.

The use of HPLC with different detectors has been investigated for the analysis of bile acid impurities present in four different commercially available ursodeoxycholic acid preparations. The bile acids were efficiently separated by C18 reversed-phase HPLC using methanol-water (3:2, v/v) as the mobile phase. The detectors used for bile acid detection were: UV at 200 nm refractive index (RI) and an evaporative light scattering mass detector (ELSD II). A prederivatization method with the formation of a fluorescent naphthacyl ester has also been used. GC-MS analysis of Me-TMS bile acid derivatives was included as a reference method. The four ursodeoxycholic acid samples were 98-99% pure. The main impurities present in the samples were chenodeoxycholic acid and to a lesser extent lithocholic acid. Only one sample was found to be almost 100% pure using all the detectors. Significant agreement of the data was found between RI, ELSD II detectors and the fluorescent method; the UV detector was unsuitable for use in this method. The analytical performances of the four detectors for bile acid analysis are reported and discussed. When the four-detector data were compared with the GC-MS method, reasonable agreement resulted. Discordant results were found in the quantitation of trace impurities like lithocholic acid and/or other minor bile acids present in amounts less than 0.1%.

Bile Acids and Salts↗

Three-dimensional confocal microscopy and visualization of the in situ cornea.

The in situ cornea is an ideal test specimen to evaluate techniques for 3D reconstruction and visualization of unstained, unfixed, transparent living tissues from a stack of optical sections. The 0.4 mm thick transparent specimen has been optically sectioned into 365 sections using a confocal laser scanning microscope (CLSM) with a water immersion objective. Depth-dependent light attenuation due to absorption and scatter within the specimen was manually compensated at each sampled section. A water immersion microscope minimized the spherical aberrations that would have occurred with the use of an oil immersion objective. Isometric sampling resulted in near-cubic voxels, which compensated for the reduced microscopic resolution in the z axis as compared to x and y resolution.

Animals↗

Simple monochromatic refractometer for trans-epidermal water loss (TEWL).

We have developed a novel, portable refractometer (Noevir-Eva) for measuring transepidermal water loss (TEWL) which consists of a light emitting diode and a phototransistor. Cobalt chloride is used as an indicator of the amount of water. Filter paper, previously soaked with cobalt chloride solution and then dried, is attached to the skin with tape (3M Blenderm). Then, the monochromatic light (670 nm) which is yielded from the light-emitting diode is irradiated, reflecting on the above-mentioned filter paper, and the intensity of the reflected light is detected by a phototransistor. The amount of water evaporation from the surface of the skin is determined by the unit of reflected light intensity. The measured values by this new apparatus show a linear relationship with those of the commercially available apparatus (Evaporimeter EP-1). Accordingly, this new refractometer for the TWEL proved to be sensitive, dependable and also inexpensive.

Adult↗