Image J was recommended to me as a possible tool for measuring fluorescence in impinging sheets of liquids. Impinging sheets are similar to impinging jets, but instead of jets the thickness for each sheet at impingement is about the thickness of a piece of printer paper. When the sheets collide, energy is release and mixing occurs very quickly. I want to watch a fluorophore in one of the liquids disappear when it encounters a quencher (or alternatively a fluorescent titration of base with acid). The attached picture is a very old picture but shows two sheets impinging. There is a pretty well defined impingement zone line. The sheet in the front is 0.01 M sodium hydroxide with coumaric acid, which fluoresces in base. The sheet in the back 0.012 M hydrochloric acid. Both sheets have the same flowrates. When the two sheets impinge the acid neutralizes the base and the coumaric acid reverts to the non-fluorescent form. Since this is a diffusion-limited process, I am hoping to be able to measure the distance from the impingement zone to where the fluorescence is very low. I know the velocity of the sheets, so I can calculate the mixing time. I have two questions for now (I know I don’t know enough to ask more questions):
- How do I determine the concentrations? I will know the concentration of acid and base prior to impingement. When determining concentration, do I need to substract the grayscale reading for the background of this photo (which is around 26) from a reading at any point in the mixed sheet? For example, point A on the photo.
- If I draw a line from above the impingement zone to below the impingement zone, there is a peak that occurs in the impingement zone as shown by plot profile. Since the base is diluted by a factor of two (each sheet has the same flowrate), why would the fluorescence intensity go up? Is it due to turbulence in the impingement zone?
There are two potential complications:
Because the sheets start from a theoretical point origin and spread out in a fan shape, the sheets thin with distance from the origin. I suspect the fact that the sheet gets thinner will affect the fluorescence intensity even if the concentration of the active form of coumaric acid does not change. Could someone confirm this.
If turbulence impacts the intensity in the impingement zone, will it also affect the intensity in the mixed sheet. The turbulence dies out rapidly, but is the drop in intensity just beyond point A due to neutralization of the base or turbulence having died out or both.
Thanks for any help to get me started. Image-J could be a big, big help with my experimentation if I can figure out how to use it for this purpose.