Separation of nanorods by density gradient centrifugation

The experimental conditions necessary for the synthesis of well-defined nanoparticles are often difficult to control. There is thus a compelling need for post-synthesis separation of nanoparticles polydispersed in size and shape. We demonstrate here both theoretically and experimentally that gold nanorods with diverse aspect ratios can be separated using density gradient centrifugation. By analysing the force balance of a Brownian rod falling in a Stokes flow, we derive a rigorous and predictive model that reveals the quantitative dependency of the nanorod sedimentation rates on their mass and shape. The calculations show that while mass dependency is still the dominating factor during centrifugation, the shape factor is not insignificant. Relatively heavier but long and thin rods could sediment slower than certain size of lighter spheres, and some rods and spheres with different masses and shapes may never be separated. This mass and shape dependency is exploited to separate as-prepared gold nanorod colloids by sucrose gradient centrifugation. Two layers of nanorods with narrow aspect-ratio distributions are obtained.     

Numerical analysis of density gradient centrifugation profiles from eukaryotic DNA

A numerical method for the deconvolution of superimposed Gaussian distributions with a unique solution has been proposed by Medgyessy [10]. We have tested the usefulness of this method for the analysis of density gradient centrifugation profiles from eukaryotic DNA, which are normally composed from overlapping Gaussian distributed profiles of several subcomponents with different mean buoyant densities. From the analysis of human DNA and from model calculations we conclude that major subcomponents can be identified by this method, if they differ in their buoyant density by approximatly 0.005 g/ml. Minor components can only be identified if the total DNA has been fractionated according to buoyant density and the analysis is performed on the density profiles of the subfractions. This procedure represents a quick method to determine a reliable minimum number of subcomponents of DNA.This paper was presented at the VI. Symposium on Analytical Ultracentrifugation, Marburg, FRG, February 16–17, 1989.     

Static density gradient centrifugation; extended Hermans-Ende equation

An extended Hermans-Ende equation for real solutions has been derived in order to calculate the density as a function of the radial distance in a density gradient mixture (light/heavy medium) inside an ultracentrifugal cell. The equation has been tested with equilibrium density gradient measurements of the system water/metrizamide/polystyrene latex where metrizamide is the heavy medium. The solvent compositions were 8 to 15 mass-% metrizamide (2-(3-acetamido-5-N-methylacetamido-2,4,6-triiodobenzamido)-2-deoxy-D -glucose). The latex concentration was 0.03 mass-%. It is demonstrated that the density gradient is highly influenced due to monideality effects with respect to the solvent composition and due to the pressure gradient in the ultracentrifuge.     

Facile fabrication of gradient density organic aerogel foams via density gradient centrifugation and UV curing in one-step

Journal of Sol-Gel Science and Technology - Gradient density porous materials have played an important role in areas of aerospace, biomaterials and high-energy absorption experiments. In order to...     

Effects of soluble fractions from untreated and SiO2-treated subcellular particles of macrophages on nucleic acid metabolism in isolated nuclei of experimental granulation tissue.

Nuclei isolated from proliferating granulation tissue were incubated with 20 000 g supernatants from untreated and SiO2-treated subcellular particles of rat peritoneal macrophages in the presence of radioactive nucleic acid precursors. The supernatant from SiO2-treated subcellular particles increased the incorporation of [3H]CTP into nuclear RNA maximally by 26% at 5 min, and that of [methyl-3H]dTTP into DNA by 16% at 20 min. The release of radioactivity from labeled DNA was suppressed simultaneously. An RNase preparation from rat peritoneal macrophages enhanced the release of radioactivity from labeled DNA similarly as the soluble fraction from untreated subcellular particles of macrophages. The results suggest that the effects of the soluble fractions upon DNA metabolism of granuloma cells are at least partly independent of the effects on RNA metabolism and that the soluble fraction from SiO2-treated subcellular particles of macrophages stabilizes DNA through inhibition of nuclease activity.     

Determination of phospholipid phosphorus in serum lipoprotein fractions obtained by density gradient ultracentrifugation

Analytical and Bioanalytical Chemistry -     

Preparation of monodisperse silicon nanocrystals using density gradient ultracentrifugation

We report the preparation of monodisperse silicon nanocrystals (ncSi) by size-separation of polydisperse alkyl-capped ncSi using organic density gradient ultracentrifugation. The ncSi were synthesized by thermal processing of trichlorosilane-derived sol-gel glasses followed by HF etching and surface passivation with alkyl chains and were subsequently fractionated by size using a self-generating density gradient of 40 wt % 2,4,6-tribromotoluene in chlorobenzene. The isolated monodisperse fractions were characterized by photoluminescence spectroscopy and high-angle annular dark-field scanning transmission electron microscopy and determined to have polydispersity index values between 1.04 and 1.06. The ability to isolate monodisperse ncSi will allow for the quantification of the size-dependent structural, optical, electrical, and biological properties of silicon, which will undoubtedly prove useful for tailoring property-specific optoelectronic and biomedical devices.     

One-step subcellular fractionation of rat liver tissue using a Nycodenz density gradient prepared by freezing-thawing and two-dimensional sodium dodecyl sulfate electrophoresis profiles of the main fraction of organelles

In the present study, we describe a new procedure using freezing-thawing to density gradient solution of Nycodenz for one-step separation of organelles from the rat liver and subsequent proteome analysis of subcellular fractions. To prepare two-dimensional electrophoresis (2-D PAGE) profiles of tissue organelles, we performed one-step subcellular fractionation of rat liver homogenate using a density gradient of Nycodenz solution, which resulted in the separation of the cytosolic fraction from the postnuclear supernatant. The density gradient of Nycodenz was prepared from a 20% solution in a centrifuge tube by freezing-thawing overnight at -20 degrees C and at room temperature for a few hours without the initial centrifugation procedure. The shape of the gradient density curve was dependent on Nycodenz concentration and tube size. After fractionation, the protein profiles were examined using one-dimensional sodium dodecyl sulfate (SDS)-PAGE. The organelles were confirmed using Western blotting. Our results indicate that our procedure provides a simple method for the separation of organelle fractions from the rat liver tissue.     

Lectin-induced retardation of subcellular organelles during preparative density gradient electrophoresis: selective purification of plasma membranes

Plasma membranes (PM) are difficult to separate by conventional means from other cellular compartments. Using a density gradient electrophoresis (DGE) apparatus (7 cm, x 2.2 cm), mammalian subcellular organelles were separated from a total postnuclear supernatant. The sialic acid-binding lectin wheat germ agglutinin (WGA) permitted 1.5-fold electrophoretic retardation of plasma membranes lagging far behind endoplasmic reticulum, endosomes, Golgi and lysosomes (in order of increasing electrophoretic mobility). Mobilities of the latter organelles were not affected by wheat germ agglutinin. The retarded plasma membrane was monitored by surface iodination, the presence of Ca(++)- and Na+/K(+)-ATPases and by the presence of clathrin-coated pits using Western immunoblotting. In the presence of WGA two clathrin-containing compartments were detected; in the absence of WGA three clathrin populations were seen in the electropherogram: clathrin-coated vesicles, clathrin-coated pits (on the PM) and clathrin-coated structures on the trans-Golgi network (TGN). Both in the presence and absence of WGA, plasma membrane domains of different electrophoretic mobilities were detected.     

Potentiometric-spectroscopic evaluation of metal-ion complexes by humic fractions extracted from corn tissue

Humic fraction (HF) functional group-type and content are expected to depend on molecular size, which in turn, is expected to influence formation of heavy-metal complexes. In this study, corn (Zea mays L.) stalks and leaves were decomposed for an 8-month period to produce water-soluble humic substances. These substances were separated into three water-soluble fractions, HF1, HF2 and HF3, from highest to lowest relative molecular size. Functional group determination showed that carboxylic, and phenolic OH acidity increased as relative molecular size of humic fractions decreased. We also observed decreasing C/O ratios from larger to smaller corn tissue-derived humic fractions, whereas N/C and H/C ratios remained relatively unaffected. Furthermore, using potentiometric titration and FTIR spectroscopy we studied formation of Ca2+-, Cd2+-, and Cu2+-humic fraction complexes and how they were affected by pH and molecular size. We determined that metal-humic complexes exhibited at least two types of functional group-sites with respect to Ca2+, Cd2+, and Cu2+ complexation. Strength of metal-ion humic complexes followed the order Cu2+ > Cd2+ > Ca2+ and was affected by pH, especially for low affinity sites. Carboxylic groups were most likely the dominant group-sites involved in complex formation. Magnitude of the metal-humic formation constants in the logarithmic form at the lowest equilibrium metal-ion concentration, under the various pH values tested, varied from 5.39 to 5.90 for Ca2+, 5.36 to 6.01 for Cd2+, and 6.93 to 7.71 for Cu2+. Furthermore, the formation constants appeared to be positively influenced by decreasing molecular size of water-soluble humic fraction, and increasing pH. However, our molecular spectra showed that the pKa of corn humic fractions increased with decreasing relative molecular size and that Cu2+ was more covalently bonded by humic fractions than were Ca2+ and Cd2+, and the nature of the covalent bond character was independent of pH.     

Determination of carbon nanotube density by gradient sedimentation

Density gradient centrifugation is a high-resolution technique for the separation and characterization of large molecules and stable complexes. We have analyzed various nanotube structures by preparative centrifugation in sodium metatungstate-water solutions. Bundled, isolated and acid-treated single-walled nanotubes (SWNTs) and multiwall nanotubes (MWNTs) formed sharp bands at well-defined densities. The structure of the material in each band was confirmed by transmission electron microscopy and Raman spectroscopy. Our data suggest respective densities of 1.87, 2.13, 1.74, and 2.1 g/cm(3) for bundled, isolated, and acid-treated SWNTs and MWNTs. These measured results compare well with their calculated densities.     

Properties of plasma membranes from granulation tissue with reference to extracellular matrix.

Treatment with neuraminidase decreased the activity of Na+,K+-activated Mg2+-adenosine triphosphatase in plasma membranes isolated from experimental granulation tissue but not that of 5'-nucleotidase or leucine-beta-naphthylamidase. A temporary lowering of the pH of the plasma membrane suspension to 2-3 inactivated all three enzymes, which remained inactive after the pH had been readjusted to 7.4. Addition of dextran preparations to the membrane suspension decreased the activity of adenosine triphosphatase. Ethanol (0.4%) had a similar effect. These marker enzymes of plasma membranes were not affected by additions of hyaluronate, chondroitin sulfate, protein polysaccharide or soluble collagen. Serotonin stimulated the adenosine triphosphatase activity slightly. About 10-20% of the protein in the plasma membrane preparation was extracted with EDTA. This "fuzzy coat" fraction yielded a distinct gel-electrophoretic protein pattern. Hyaluronidase was not helpful in cleaving this surface layer from the plasma membranes.     

Chemical heterogeneity in poly [styrene-co-(butyl methacrylate)] copolymer latexes prepared using different monomer addition modes. A study by isopycnic centrifugation in density gradient

 Three different styrene-butyl methacrylate copolymer latexes were prepared by a uniform procedure but introducing styrene (S), butyl methacrylate (BMA), and minor amounts of acrylic acid (AA), in three different orders: i) simultaneous monomers addition, which yielded {P(SBMA)}; ii) addition of S (and half of the AA) followed by BMA (and the remaining AA), yielding {PS/PBMA} and iii) the inverse order, {PBMA/ PS}. Product characterization was done by centrifugation in density gradients coupled to scattered light scanning photometry of the centrifugation tubes. IR and NMR spectra were obtained from bulk polymer as well as from isopycnic centrifugation fractions. In agreement with findings of other authors, the particles produced by simultaneous monomer addition {P(SBMA)} are made out of the statistical copolymer, whereas sequential monomer addition leads to the formation of latex with homopolymer domains. IR and NMR spectra of {PS/PBMA} and {PBMA/PS} are identical but isopycnic density band profiles of all three samples are distinct. Acrylic acid residues are not detected in the dialyzed latex, using both IR and NMR. Spectra of latex isopycnic fractions do also show significant differences arising from their monomer chemical compositions, but isopycnic centrifugation and spectral data do not reveal any correlation between particle density and monomer composition. Isopycnic centrifugation can thus solve two problems on latex characterization: first, it is a high-resolution preparative technique, unmatched by any other separation method. Second, it yields latex particle fingerprints, which are dependent on particle chemical characteristics, rather than on particle diameters. Received: 19 March 1996 Accepted: 29 August 1996     

Calculation of some atomic properties by gradient corrected density functional method

Using thePerdew andPerdew-Wang Yue gradient expansion functionals the total electronic binding energies, the ionization potentials and the KL transitin energies are calculated for the atoms of elements from Be to Cu, and the 3d–4s transition energies are determined for the third row elements. There is a general improvement with respect to the results found without gradient corrections. However the asymptotic behavior of the potential does not changes.