# Fluorescence pH Curve

Classroom Exercise

There is an Excel spreadsheet for the class data and an Excel spreadsheet for the professor’s data.  You will need to use your computer to do the statistics to complete the questions.

In this example: each student in the class made a solution of salicylic acid at a different pH.  They then pipetted 300 uL of their solution into a 96 well plate, doing duplicate (i.e. 2 wells) per person (so in the spreadsheet B1&C1&D1 are the same, B2-C2-D2 etc).  The fluorescence of the solutions (in arbitrary fluorescene units) were then collected with an excitation wavelength of 299 nm and an emission wavelength of 410 nm.

1. Average the three replicates for each person.

2. Calculate statistics on the data set to determine if the fluorescence varies by pH value. What statistics did you do and why? What was the conclusion?

3. In your Excel spreadsheet make a graph using the student’s data of fluorescence intensity vs pH. Why does the graph have that shape? Sketch the shape of the graph below and estimate and mark where the pKa is for the salicylic acid.

4. The best way to actually find the pKa is to do a first derivative plot. The first derivative is Δ(fluor intensity)/Δ pH. Δ is the change between two values (i.e. subtraction).  You then plot that as the y-value vs the average of the pH unit.   Make a first derivative plot of the student data.  The peak will be the pKa value (i.e. the midpoint on the graph before).

5. The professor also did this titration and ran it in the plate reader. Because she did it on a different day and with different settings, her values can’t be averaged with yours. However, take her data and plot the fluorescence intensity vs. pH and the first derivative plot.  How do the professor’s graphs compare to the student’s answer?  What pKa does the professor get?

1. Why does the fluorescence of salicylic acid change with pH? This article will be a good reference you out: Kort et al. BMC Biotechnology 2010, 10:45 http://www.biomedcentral.com/1472-6750/10/45

2. What does the article say the pKa of salicylic acid is? How does that compare to the professor and student answer?

3. How were scientists in the article using the change in salicylic acid fluorescence to do a biological experiment?