I am trying to measure the absorption spectrum of a photo-caged fluorescent probe on an Agilent Cary 100 UV-Vis. I tried a 10mM solution in DMSO after a DMSO blank and the spectra had a broad flat peak from ~300-600 nm. I know this is wrong because the fluorophore on the probe is TAMRA which should have a high peak around 557nm. Is this happening because the 10mM concentration is too low or is there something else I'm not doing correctly?
The concentration was way too high. The concentration should be adjusted so that the peak absorbance is in the 0.3 to 1 range. The extinction coefficient of TAMRA is 90,000 M-1cm-1. If you are using a cuvette with a 1 cm pathlength, the concentration of TAMRA should be no more than 10 micromolar.
Leslie, a broad flat peak from ~300-600 nm indicates that your concentration is way too high. As pointed out by Prof. Shapiro, you need to dilute your 10 mM solution 1000x to do the measurement.
Hi Leslie Spitalny as a general strategy, you can use 10 uM solutions for absorption measurements.
The concentration of sample is required to measure the absorption spectrum on a UV/Vis Spectrophotometer is the range 1x10-4 Moles.
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