User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2013/09/03: Difference between revisions
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Revision as of 10:11, 29 August 2013
 Biomaterials Design Lab
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ObjectiveToday we'll be determining the molar absorptivities of two different molecules, adenosine and inosine. The data that we generate today will be important when we study adenosine deaminase (ADA), which converts adenosine to inosine. The difference between these two molecules is that adenosine contains a primary amine whereas inosine contains a carboxy group. Overexpression of this protein causes anemia in humans. A shortage of this protein can lead to severe immuno-defficiency. Adenosine and inosine have different absorption spectra. We will be observing changes in UV-Vis spectra to determine changes in concentration of both adenosine and inosine. In order to do this, we will need to know the molar absorptivity (ε) of both of these molecules. Just as each molecule has a characteristic absorption at each wavelength, this (per-wavelength) absorption can be quantified by a molar absorptivity. Or ... for a given concentration a molecule will absorb a very specific amount of light at a precise wavelength. A molecule doesn't have just one molar absorptivity; there is a molar absorptivity to describe each wavelength in a molecular absorbance spectrum. DescriptionIn order to determine ε for any substance (molecule, protein, gold nanoparticle), you need to determine how the absorbance of that substance changes with concentration. Taking our Beer's Law relationship: (The darker the beer, the stronger the brew!) A=εbc where b is the path length (1cm), we can see that A is directly proportional to c and that if we plot A vs C, the slope will be ε. That will be your task for the day. To determine molar absorptivity of both adenosine and inosine by plotting graphs of A (at one specific wavelength; I suggest you use a peak value from the spectrum) vs c. We are also going to pool data from all of the groups to develop a full calibration curve. We are going to determine standard deviations from the group's data, determine a confidence interval, and perform a Q-test to remove any outlying data. DirectionsStock Solutions You are going to need to make two stock solutions, one for each molecule. These stock solutions should be prepared such that you can make the following solutions: NotesThis area is for any observations or conclusions that you would like to note.
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