Why do fluorescence intensities decline with replicate number? 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?
That looks like a bleaching to me. The notion that bleaching only occurs with excesive light intesity on small populations of fluorophores is news to me. Might also that in my field we just use the bleaching term loosely. Best way to test is to take a long exposure of your sample and monitor the fluorescence intensity over time. 
With our chemistry we usually encounter intensity loss that can be fitted with exponential decay function which would explain the bottoming out that you are describing. We often take multiple such "bleaching curves" and fit them to correct for intesity loss in our experiments that can take multiple hours.
In case you are only exposing your sample for short periods i would just make sure you measure each sample the same number of times that should rule out any error between samples. Thus reducing to only one measurment will not make it more correct.
Bleaching is poorly understood thus far. There is not a way to predict what excatly happens with a specific molecule. 


Also how big is the effect for example (Start value)/(3rd measurment value) ?