Meet (or Re-Meet) Fluorescein

http://en.wikipedia.org/wiki/Fluorescein

http://en.wikipedia.org/wiki/Fluorescein

“The fluorescence of this molecule is very high, and excitation occurs at 494 nm and emission at 521.”

Absorption and Emission of Light

From: Molecular Probes

http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/pH-Indicators/Probes-Useful-at-Near-Neutral-pH.html#head1

Obtain fluorescein sample from TA; blank with appropriate buffer & re-blankwhen you switch to a different pH. Determine appropriate dilution to use, and scan from 200 – 600 nm. Move cursors to obtain absorbance values at a particular lambda max.

Analyze your data using

(1) Nonlinear fitting

(2) Linear fitting

(3) 2nd Derivative fitting

To speed things up, we will read at 240 & 490 nmrather than scanning from 200 – 600 nm.