User:Daniel-Mario Larco/Notebook/AU Biodesign Lab - 09/03/2013/2013/09/03
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Today 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.
In 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!)
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.
You are going to need to make two stock solutions, one for each molecule. Adenosine has a molecular weight of 267.24g/mol; inosine has a molecular weight of 268.2g/mol. You should confer with your group members how you want to prepare these stock solutions and prepare sample calculations for dilutions. (That is ... come in prepared to make these! I don't want people discussing for 30minutes how they want to do this.) I These stock solutions should be prepared such that you can make the following solutions:
- The stock solution for adenosine was made from 0.1011g of adenosine in 100mL of water, to give a concentration of 0.003783 M. - The stock solution for inosine was made from 0.103g of inosine in 100ML of water, to give a concentration of 0.003840 M. - Each of the sample solution were made from these stock solutions. The volumes of stock solution used for each sample was determined by the equation M1v1=M2v2.
Absorption Spectra Each group will have a cuvette to work with. Take a spectrum of each of your samples along with a blank. Be sure to be considerate of everyone who wants to use the spectrometer. You will need to rinse your cuvette between each sample. Within each group, I want you to be prepared for how you are going to do this as well. Not everyone needs to be filling cuvettes, taking spectra, cleaning cuvettes, converting data. Talk among yourselves for how your group is going to handle the data collection and analysis. You're working in teams ... people should have roles (even if you are rotating samples, with one person collecting the data for sample A, then another person taking sample B, etc)! As you are collecting data, you NEED to be importing it into Excel and correcting it (subtracting the blank spectrum).
- Here are the absorption spectra for adenosine.
- Here are the absorption spectra for inosine.
This area is for any observations or conclusions that you would like to note.