This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Handling and preparation of vaccine samples for injection All surfaces around the working area were disinfected with 75% isopropanol prior to start of the experiment. Upon opening the packet containing the vaccine samples, each vial was disinfected externally by rolling in a 75% isopropanol, thawed at room temperature, and suspended in near boiling water (not immersed completely) for at least 10 min to inactivate the virus. All vials used for injections were disinfected externally with 75% isopropanol prior to loading in the machine. About 40 ?L of each vaccine was obtained from respective original container, transferred into vials and injected to observe initial concentration of sugars of interest. Peaks of sugar from each vaccine initial concentration were too large and can not be quantified. Hence, each vaccine sample was diluted 300 times prior to injection in the machine. Three replicates from each vaccine (300 times dilution) were prepared and injected individually. At the end of the analysis, all materials, glass wares, excess samples prepared, waste were autoclaved and disposed properly. Composition analysis by HPAEC The samples were analyzed for glucose and sucrose composition by High pH-Anion-Exchange Chromatography (HPAEC). A mix of standards (glucose and sucrose) with 4 known concentrations (0.5, 1.0, 2.0, and 4.0 nmoles per injection) were prepared to establish a calibration equation. The number of moles of each sugar in the vaccine sample was quantified by linear interpolation from the calibration equation. The sugars were analyzed using a Dionex DX500 system equipped with a GP40 gradient pump, an ED40 electrochemical detector, and a Thermo-Separation AS3500 autosampler containing a stainless steel needle. Glucose and sucrose were separated by a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an amino trap. The gradient programs used the following eluents: A, water and B, 100 mM NaOH. Autoinjections were made every 20 min and the autosampler was set to deliver 10 ?L of sample or standard solution per injection. Blanks (water) were injected between standards and the samples to prevent carry-over contamination. Instrument control and data acquisition were accomplished using Dionex PeakNet software, version 5.01.
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