I am performing a determination of riboflavin with fluorescence spectrophotometry. I've dissolved my riboflavin in a dilute GAA solution, created a calibration curve and measured my unknown's fluorescence.
For these types of analyses I usually perform triplicate measurements, however I've noticed that spectrofluorometric measurements always decrease with replicate number. I assume this is due to 'photobleaching' of my samples. Since the phenomenon occurs with replicate rather than over time, and since my sample is in a dark environment, I would imagine this can only be the result of the excitation light emitted by the instrument itself. I find it surprising that this causes such a dramatic change in fluorescence from measurement to measurement. I also notice that with enough replicate measurements, eventually the intensity decline seems to bottom out. Can anyone explain the specifics of this phenomenon to me?
- Am I right in assuming that this is photobleaching (deactivation of the chromophore) rather than photodegredation (breaking the molecule), or are these the same?
- How exactly is the fluorophore deactivated?
- Can the degree of fluorescence intensity decline be predicted quantitatively?
- Would my calibration be more accurate if I used single measurements instead of taking the mean of three replicates?