User:Andrew K Farag/Notebook/Andrewfarag/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 G-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:
For the RANDOM sample, I want each group to make an unknown (it will be known inside of the group, but not outside of the group) for each molecule. I want a different group to see if they can determine the calculation of your unknowns from the calibration curve that we put together as a class.
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).
Calibration Curve and Group work As a large group, determine what wavelengths you want to use for your adenosine and inosine calibration curves (A vs c). Choose two people (one for each molecule) to compile your A(λ) and concentration data from each group. Do a least squares fit to the data and determine the slope of the line (remember the intercept should be zero --- with a concentration of 0 there should be no absorbance). This data, once compiled should be shared with all of the group members (via dropbox).
Determine the standard deviation for your data points.
Determine the confidence interval for 90% and 95% confidence.
Determine if any data can be ruled out using a Q-test.
Unknown Groups should exchange unknowns and try to determine the concentration of these unknowns from the calibration curves. In a week, I want you to revisit this data and propagate the error from the calibration curve to your concentration calculation. After making your calculation, find out from the group, whose unknown you are using, what the calculation of their samples should be.
To Dilute the solution of Adenosine and Inosine to the concentrations listed above, the following equation is used.