PMC:2812708 / 7331-10574
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2812708","sourcedb":"PMC","sourceid":"2812708","source_url":"https://www.ncbi.nlm.nih.gov/pmc/2812708","text":"Materials and methods\nThe β-carotene product was obtained in powder form as a laboratory sample from BASF SE, Ludwigshafen. An aqueous dispersion in water was prepared with a concentration of 0.05 g/l. The UV/vis spectrum of the dispersion and of the free gelatin is shown in Fig. 1.\n\nMulti-wavelength analytical ultracentrifugation (MWL-AUC)\nA Multiwavelength AUC as described in Bhattacharyya et al. (2006) and Strauss et al. (2008) was used at 25°C. We have applied a band centrifugation experiment using a Vinograd cell. In contrast to conventional sedimentation velocity experiments, where the sample between boundary and bottom of the cell is only diluted by radial dilution, the sample in band centrifugation is diluted additionally by fractionation in the pure solvent. The reservoir is filled with a small amount of concentrated sample. Column and sample sectors are filled with D2O with a density which is higher than that of the sample solution and lower than the density of the dispersed solute. We have prepared a 20 g/l solution and deposited 15 µl of the solution into the cell reservoir. After preliminary experiments, this concentration was chosen to ensure that the individual components of the mixture are detected in as many as possible scans with OD’s \u003c 1.4 in the experiment without too much dilution which causes noisy data.\nAfter cell assembly, the AUC was accelerated to 5,000 rpm for 3 min to transfer the sample in the reservoir via capillaries to overlay the D2O column. Afterwards, the speed was increased to 55,000 rpm. Forty scans were taken with a time interval of 90 s and a radial step size of 50 μm to observe the full sedimentation of the sample. The selected wavelength range was 250–750 nm. In the prototype setup, we apply the spectrum acquired for an empty cell as a reference for the calculation of the absorption leading to a baseline offset of 0.05 OD (see Fig. 3i) After the experiment, while cleaning the cell, we saw some precipitate in the cell reservoir. Thus, not all particles were transferred to the sample column, but some big particles remained in the reservoir as they already must have completely sedimented upon speeding up the rotor to 5,000 rpm.\nFig. 3 Three-dimensional plots of the raw data from a band sedimentation experiment with β-carotene detected with the MWL detector. The axes are wavelength, absorbance and radial position. i Scan 1 (1.5 min); ii Scan 10 (15 min); iii Scan 18 (27 min); iv Scan 40 (60 min)\nEach of the 40 scans produces a 3-dimensional graph. We have radial position as x-dimension, wavelength as y-dimension and absorbance as z-dimension. In the present contribution, we will perform a semi-quantitative evaluation based on simple model-free transformations of the data without any prior knowledge.\nFor evaluation, we have converted the radial position (r) to the sedimentation coefficient s by using Eq. 1. In Eq. 1, r m is the radial position of the meniscus and ω2 t is the run time integral.1\nThe 3D absorption dataset can now be projected either onto the wavelength or sedimentation coefficient axis to better visualize the spectral changes with different sedimentation coefficients or sedimentation coefficient distributions at different wavelengths.","divisions":[{"label":"title","span":{"begin":0,"end":21}},{"label":"p","span":{"begin":22,"end":283}},{"label":"title","span":{"begin":285,"end":342}},{"label":"p","span":{"begin":343,"end":1347}},{"label":"p","span":{"begin":1348,"end":2475}},{"label":"figure","span":{"begin":2204,"end":2475}},{"label":"label","span":{"begin":2204,"end":2210}},{"label":"caption","span":{"begin":2211,"end":2475}},{"label":"p","span":{"begin":2211,"end":2475}},{"label":"p","span":{"begin":2476,"end":2785}},{"label":"p","span":{"begin":2786,"end":2983}},{"label":"label","span":{"begin":2982,"end":2983}}],"tracks":[{"project":"MyTest","denotations":[{"id":"19242689-19816525-28778239","span":{"begin":429,"end":433},"obj":"19816525"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"attributes":[{"subj":"19242689-19816525-28778239","pred":"source","obj":"MyTest"}]},{"project":"2_test","denotations":[{"id":"19242689-19816525-28778239","span":{"begin":429,"end":433},"obj":"19816525"}],"attributes":[{"subj":"19242689-19816525-28778239","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"MyTest","color":"#93ecae","default":true},{"id":"2_test","color":"#ec9493"}]}]}}