Fractionation of soybean phospholipids by preparative high-performance liquid chromatography with sorbents of various particle size.
Normal-phase high-performance liquid chromatography was used on a preparative scale to seperate phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylcholine (PC) phospholipids from soybean. Separation was achieved using mixtures of three solvents, hexane, methanol and isopropanol. The optimized mobile phase compositions were experimentally determined while operating in a linear gradient mode using 15, 5-20, 25-40, and 40-63 microm preparative particles as well as 4 microm analytical particles. A gradient mobile phase was established on a commmercially available analytical Nova-Pak column such that hexane linearly decreased from 85 to 0 as isopropanol and methanol linearly increased in two gradient steps from 10 to 30 and 5 to 70 respectively. The total run time was 25 min at a flow-rate of 1.5 ml/min. A slight change in mobile phase composition was required to increase the resolution of phospholipids. The 15 microm particle size gave the best separation of the preparative particle sizes examined based on their resolutions between PE and PI and PI and PC. Finally, the retention factors of PE and PC were correlated in terms of mobile phase composition.
MATERIALS AND MODIFICATION OF ELECTRODES FOR THE DETECTION OF BIOLOGICAL MOLECULES
MATERIALS AND MODIFICATION OF ELECTRODES FOR THE DETECTION OF BIOLOGICAL MOLECULES by Michelle Marie Wandstrat Sol-gels have been utilized in many applications d ue to many desirable properties. A limitation of sol-gels is the gradual collapse of their structure with time. To address this problem, sol-gels were formed from tetramethoxysila ne in the presence and absence of generation-zero poly(amidoamine) dendrimers (G0-PAM AM). To test the hypothesis that G0-PAMAM, which serves as a crosslinking agent, wil l stabilize the structure, the pore volume was monitored over a 52-week period. The pre mis was that the crosslinking will result in rapid densification. Nitrogen adsorption data stabilized after 25 weeks when G0PAMAM was present. In its absence, the total pore v olume continued to decrease over the entire period. Phospholipids (PLs) determination is important in the food and pharmaceutical industries. Methods were developed for the detectio n of a PL, phosphatidylcholine (PC). Electrochemical oxidation of PC, was accomplished a t a 4-aminothiophenol (ATP)modified gold electrode coated with a layer-by-laye r assembly of electrochemical catalyst (dirhodium phosphomolybdic acid), a trapping agent for PC (a cyclophane, CP, derivative, 1,4-xylylene-1,4-phenylene-diacetate), and a spacer (generation-4 polyamidoamine dendrimer, PAMAM). The hypothesized process was verified by quartz crystal microbalance measurements which showed that Au | ATP | CP | PAMAM | CP trapped (1.5 ± 0.4)·10 -9 mol cm of PC. The oxidation of PC at 0.16 V vs. Ag | AgCl yielded a current that varied linearly with concent ration over the range 1 50 μM. Finally, an electrochemically assisted method for d eposition of nm-scale films of porous silica was developed. In aqueous, un-buffere d solution at a potential where H + is generated in a tetraethylorthosilicate sol, nm-scal e, insulating deposits of silica on electrodes are formed. Inclusion of β-cyclodextrin in the sol provides conductivity, presumably by producing channels in the film. With the addition of a mediator, Rh , the electrochemical oxidation of PC is demonstrated; th e process occurs without the passivation that otherwise precludes the voltammetr ic determination of PLs. Voltammetric current proportional to PC concentrati on in the range 1 -100 μM is achieved with this electrode. MATERIALS AND MODIFICATION OF ELECTRODES FOR THE DETECTION OF BIOLOGICAL MOLECULES
Reversed-phase high-performance liquid chromatographic separation and mass detection of individual phospholipid classes.
A reversed-phase high-performance liquid chromatographic method is described for the separation of individual classes of phospholipids into subfractions. The separation was achieved with isocratic elution using a Nucleosil-5 C18 column and a mobile phase consisting of methanol, acetonitrile and water. Three commercial C18 packing materials were evaluated before Nucleosil was chosen as the stationary phase. The compounds were detected with a combination of an UV detector and a light-scattering mass detector, which provided quantitative chromatograms reported for the first time. The mass detector also allowed location of the most relevant peaks of the UV chromatogram. Phosphatidylcholine and phosphatidylethanolamine species from egg, and phosphatidylcholine species from rat liver, were resolved into more than 20 peaks. The method can be applied to the separation of phospholipids from different biological sources.