I am currently doing some research regarding beta-carotene. With reference to other sources and their methodoloy for DPPH assay, I conducted my experiment but failed to find any significant peak in absorbance at 517nm using a UV-spectro.
My sample of DPPH is already dissolved to 0.1mM in methanol.
My methodology was mixing- 0.3cm3 DPPH 0.3cm3 beta-carotene (dissolved in propanone) 2.4cm3 methanol
I increased the dissolved betacarotene in increments of 0.3cm3 ( upto 1.5cm3) while simultaneously decreasing methanol also by 0.3cm3 (upto 1.2 cm3) for a total of 5 readings.
The mixture was shaken and left to sit (in dark conditions) for 30mins before readings were taken using UV-spectrometer. The UV-spectrometer was calibrated with DPPH.
While I achieved the expected colour change from purple to yellow, I did not get a significant absorbance at any reading at 517nm even when using pure beta carotene.
I suspect this may be due to the betacarotene being dissolved in propanone which perhaps may be the wrong solvent, or perhaps i am calibrating the UV-spectro with the wrong substance, however I would like to check if this procedure is incorrect.
Please advice accordingly. Much appreciated.
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