Biology Lab - Absorbance vs. Light Intensity

Question

Hello,

 

I recently performed a photosynthesis experiment regarding change in absorbance over time vs. intensity of light. 4 tubes of solutions containing chloroplasts, .1M PO4 buffer, water, and DPIP were created and then measured for absorbance using a spectrophotometer every 5 minutes for 30 minutes. In between each absorbance measurement, the tubes were subjected to different light intensities while one was kept in a dark cabinet.

 

My results were that the absorbance values of the tube left in the dark cabinet remained high but changed very little over the 30 minutes while the tubes subjected to 24 cm, 30 cm, and 49 cm light path lengths rapidly decreased in absorbance and then increased a bit by the end of the 30 minutes. 

 

I'm not sure why this happened. Why would increased light intensity cause the absorbance to rapidly decrease while the dark tube stayed relatively the same? I would have guessed higher light intensity caused higher absorbance. My lab manual says "After it accepts electrons, DPIPH2 is reduced and becomes clear... The greater the reduction of DPIP, the more clear the solution, and the greater the absorbance when measured with a spectrophotometer." I was thinking that a longer light path length would result in more electrons, thus reducing DPIP more and causing a greater absorbance, but this must be wrong. And why would the absorbance values begin to go back up at the very end?

 

Sorry that was so wordy. I'm just not sure how to connect all of this together. I would really appreciate any assistance understanding these trends.

Answer 1

Is DPIP colored?  If so, it makes sense that the absorbance would decrease with light...not increase, as the lab manual said.  It is the transmission that increases with greater reduction of DPIP, right?  

 

Clearer solution = increased transmission = decreased absorbance!  

 

Why the numbers would go back up at the end?  I'm not sure, perhaps some of the DPIP became oxidized again, giving more color and more absorbance by the tube.  Of course this only works if DPIP starts as colored.  Then as the chloroplasts function and reduce it, the color clears and absorbance decreases. (Transmission increases) 

 

The dark tube did not change much so absorbance stayed the same.  

 

Hope this helps!