Jan Vanderdeelen

Optimization of the column loadability for the preparative HPLC Separation of soybean phospholipids

A simple and rapid preparative-scale separation method was investigated in order to obtain pure soybean phospholipids. Because of technical and economical reasons, two coarse, irregular silica gels were selected. Comparing both stationary phases, a ternary mixture of hexane, 2-propanol and water yielded a different elution order of the phospholipids at analytical sample loads, in spite of the chemical similarity of these packing materials. During scaleup, it became obvious that the retention characteritics were largely influenced by the sample load, thus making the 15–35 μm RSiL inappropriate for preparative-scale separations of phospholipids. Moreover, the column loadability could be increased by controlling the flow rate. Hence, a solvent program was elaborated which enabled a column loadability of up to 2% by weight of the stationary phase. Using analytical high performance liquid chromatography, it was shown that the method proposed yielded over 90% pure phospholipids at a recovery of nearly 80%.

Simple and rapid method for high -performance liquid chromatographic separation and quantification of soybean phospholipids

Technique de CLHP a phase isocratique entierement automatisee et pilotee par ordinateur. La phase mobile est compatible avec une detection ultraviolette. Excepte pour le phosphatidylinositol, la limite de detection des phospholipides est inferieure a 0,9 μg

Partial resolution of molecular species during liquid chromatography of soybean phospholipids and effect on quantitation by light-scattering

SummaryUsing both synthetic phospholipids and soybean phosphatidylcholine (PC), it is shown that the retention of phospholipids during normal-phase chromatography is not only determined by the polar headgroup, but also by the fatty acid composition. Although fatty acid methyl ester determinations reveal that this effect only causes an enrichment of some fatty acids in subsequent fractions of the same phospholipid class, still a large influence on the peak shape is observed. As a further consequence, the evaporative light scattering detector output is greatly affected, so that accurate quantitation of phospholipid classes requires the availability of standards of the same origin.

Phospholipid composition of r-DNA hepatitis B surface antigens

Abstract The composition of the lipid matrix of the antigenic protein of r-DNA hepatitis B surface antigen (HBsAg) particles was investigated. High-performance liquid chromatography (HPLC) revealed that the lipid fraction contained mainly neutral lipids, as well as phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine. In addition, it was shown that these lipids contained primarily C16 and C18 saturated and mono-unsaturated fatty acids. Comparing these results to literature data concerning blood plasma HBsAg particles, significant differences were observed.

Particle Sizing of Liposomal Dispersions: A Critical Evaluation of Some Quasi-Elastic Light-Scattering Data-Analysis Software Programs

AbstractThe particle size of four liposomal dispersions was estimated by quasi-elastic light scattering. The data were analyzed by three different software programs. Comparing the estimated values for the average particle size and the particle size distribution, some differences became obvious. First, it was observed that the average values obtained by different calculation methods were hardly comparable, since some systematic deviations occurred. Comparing the estimated particle size distributions, the Malvern Automeasure software was shown to produce overly smoothed, broad monomodal distributions. On the other hand, the results of both the CONTIN and the Brookhaven ISDA package agreed much better with the particle size distributions estimated by image analysis of electron microscopic photographs, revealing that all samples studied were bimodal, containing small liposomes with a rather constant diameter as well as much bigger aggregates that were gradually broken down during sonication. Concerning the IS...

Monolayer adsorption of phosphate and phospholipids onto goethite

Abstract Using goethite, the adsorption behaviour of both inorganic and organic phosphate was investigated. Potassium dihydrogen phosphate was adsorbed in a monomolecular layer. In comparing different adsorption isotherms, a double Langmuir equation yielded the best results: about 33 μmol per gram of goethite was strongly fixed, whereas the remaining 14 μmol per gram was more loosely bound, as deduced from the calculated Gibbs free energy of adsorption. By subsequent washing only the latter part could be removed. Adsorption of phosphatidylcholine from apolar solvents obeyed the Langmuir law, indicating the formation of a monolayer which contained 23.9 μmol per gram. On the other hand, multilayer adsorption occurred in water, but only the innermost layer was irreversibly fixed; this monolayer was shown to influence both the charge of the goethite surface as well as its hydrophilic/lyophilic nature. Since no multilayer formation took place upon phosphatidic acid adsorption, the ionization of the phospholipids seemed of major importance. It was concluded that in sufficiently concentrated dispersions chemisorption resulted in the formation of an irreversibly fixed monolayer of phospholipids, whereas multilayer adsorption depended on the balance between repulsive Coulombic forces and attractive hydrophobic interactions. These multilayers were easily desorbed by washing the coated adsorbent.

IUPAC-NIST Solubility Data Series. 95. Alkaline Earth Carbonates in Aqueous Systems. Part 2. Ca

The alkaline earth carbonates are an important class of minerals. This article is part of a volume in the IUPAC-NIST Solubility Data Series that compiles and critically evaluates solubility data of the alkaline earth carbonates in water and in simple aqueous electrolyte solutions. Part 1 outlined the procedure adopted in this volume, and presented the beryllium and magnesium carbonates. Part 2, the current paper, compiles and critically evaluates the solubility data of calcium carbonate. The chemical forms included are the anhydrous CaCO3 types calcite, aragonite, and vaterite, the monohydrate monohydrocalcite (CaCO3· H2O), the hexahydrate ikaite (CaCO3·6H2O), and an amorphous form. The data were analyzed with two model variants, and thermodynamic data of each form consistent with each of the models and with the CODATA key values for thermodynamics are presented.

Enzymatic Activity of Cytochrome C-Oxidase Inserted into Magnetoliposomes Differing in Surface Charge Density

The role played by the surface charge density of the phospholipid coat of nanometer-sized Fe3O4 colloids (so-called “magnetoliposomes”) in the catalytic activity of beef heart cytochrome c oxidase was investigated. Screening of various binary mixtures of the anionic dimyristoylphosphatidylglycerol and the zwitterionic dimyristoylphosphatidylcholine demonstrated that the highest degree of reactivation was found in the lower negative charge range. Pre-incubation of the charged colloidal biocatalytic particles with cytochrome c induced aggregation and reduced overall enzymatic activity. The results are interpreted in terms of a different affinity of the substrate for the various membrane types and of a reorganisation of the enzyme within the membrane matrices.

Consistency issues of aqueous solubility data and solution thermodynamics of electrolytes

For the calculation of aqueous solubility of electrolytes, thermodynamic data from different sources are required. This can lead to errors if the data are inconsistent. This study reveals that some often-used semi-empirical equations for CO2 solubility and other equilibria are inconsistent with CODATA (Committee on Data for Science and Technology) key thermodynamic data. The equations were recalculated to make them consistent with CODATA. Combining standard thermodynamic data of NaCl with the Pitzer model leads to significant deviations from the accepted experimental value at 25 °C. Owing to the inadequacy of the Pitzer model at molalities exceeding 6 mol kg–1, this model leads to poor predictions of the HCl vapor pressure in equilibrium with highly concentrated aqueous HCl solutions. A long-standing inconsistency problem is related to the solubility of calcium carbonate. The main problem is disagreement on the existence of the CaHCO3+ ion pair. It is shown that the inconsistency largely disappears if it is assumed that the CaHCO3+ exists at low ionic strength, but becomes less stable at higher ionic strength.

Optimization of a chromatographic method for the gram-scale preparative fractionation of soybean phospholipids

SummaryTo enable the study of the functional properties of pure soybean phospholipids, a chromatographic method was developed for the preparative fractionation of soybean lecithin. A coarse and irregularly shaped silica gel was used as the stationary phase, whereas the mobile phase consisted of three mixtures of hexane, 2-propanol and water of increasing polarity. These solvents were included in a step gradient, formed by an isocratic pump connected to a solvent changer.To reduce solvent consumption, the procedure was optimised using a method development column. The solvent program and the sample load were shown to affect mainly the purity and recovery of phosphatidylinositol.