User:Trisha I. Ibeh/Notebook/Trisha Notebook/2013/09/03: Difference between revisions

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==Objective==
==Objective==
The molar absorptivities of two different molecules, [http://en.wikipedia.org/wiki/Adenosine adenosine] and [http://en.wikipedia.org/wiki/Inosine inosine] were determined in this lab using UV-Vis and Beer's law.  
The molar absorptivities of two different molecules, [http://en.wikipedia.org/wiki/Adenosine adenosine] and [http://en.wikipedia.org/wiki/Inosine inosine] were determined in this lab using UV-Vis and Beer's law.  
The changes in UV-Vis spectra will be observed 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. A calibration curve from the class data will be created. From this data the the standard deviation, Confidence Interval (90% and 95% confidence) will be calculated and Grubb's test will be performed to determine the outlier.
The changes in UV-Vis spectra will be observed to determine changes in concentration of both adenosine and inosine. In order to do this, the molar absorptivity (ε) of both of these molecules will be known. A calibration curve from the class data will be created. From this data the standard deviation, Confidence Interval (90% and 95% confidence) will be calculated and Grubb's test will be performed to determine the outlier.


==Description==
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!)
.


A=εbc
==Dilutions==
 
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.
 
==Directions==
<u>Stock Solutions</u>
<u>Stock Solutions</u>


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. (<b>That is ... come in prepared to make these!</b> 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:
Stock solutions were made to create these dilutions for each molecule. The calculations for the stock solution and dilutions were performed before lab.  
{|style="width:700px"
{|style="width:700px"
|<u>Adenosine solution concentrations (M)</u>
|<u>Adenosine solution concentrations (M)</u>
Line 59: Line 47:
|}
|}


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.
The RANDOM concentration was 0.25*10^-5 for adenosine and 0.4*10^-5 for inosine.


<u>Absorption Spectra</u>
'''Preparation of Dilutions of Adenosine'''
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).


<u>Calibration Curve and Group work</u>
[[Image:Screen_Shot_2013-10-05_at_4.20.59_AM.png]]<br>
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.
'''Preparation of Dilutions of Inosine'''


Determine the confidence interval for 90% and 95% confidence.  
[[Image:Screen_Shot_2013-10-05_at_3.41.48_AM.png]]<br>
The groups then exchanged unknowns to determine the concentration from the calibration curves. In a week, the data will be revisited and the error will be propagated from the calibration curve to the concentration calculation. After making the calculation, the calculation of the unknown from the group will be compared.


Determine if any data can be ruled out using a Q-test.
[[Category:Course]]
[[Category:Miscellaneous]]


<u>Unknown</u>
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.


==Notes==
This area is for any observations or conclusions that you would like to note.


[[Category:Course]]
[[Category:Miscellaneous]]


Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.
==Data==
'''Adenosine Absorbance Spectrum at 260 nm'''


[[Category:Course]]
[[Image:Screen_Shot_2013-10-05_at_5.21.54_AM.png]]<br>
[[Category:Miscellaneous]]


'''Adenosine Calibration Curve at 260 nm
'''


[[Image:Screen_Shot_2013-10-05_at_4.31.20_AM.png]]<br>


[[Category:Course]]
'''Class Calibration Curve for Adenosine at 259 nm'''
[[Category:Miscellaneous]]


[[Image:Screen_Shot_2013-10-05_at_4.43.15_AM.png]]<br>


==Notes==
Due to the time restrictions of the lab, the inosine spectra was taken the next lab (9/4/13).


Grubb's test was conducted to determine the outliers in the pooled class data.


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Revision as of 02:35, 5 October 2013

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The template for this lab can be seen from Dr. Hartings lab. Values are altered to accurately describe the lab that was conducted on this day. The template can be found here

Objective

The molar absorptivities of two different molecules, adenosine and inosine were determined in this lab using UV-Vis and Beer's law. The changes in UV-Vis spectra will be observed to determine changes in concentration of both adenosine and inosine. In order to do this, the molar absorptivity (ε) of both of these molecules will be known. A calibration curve from the class data will be created. From this data the standard deviation, Confidence Interval (90% and 95% confidence) will be calculated and Grubb's test will be performed to determine the outlier.


.

Dilutions

Stock Solutions

Stock solutions were made to create these dilutions for each molecule. The calculations for the stock solution and dilutions were performed before lab.

Adenosine solution concentrations (M) Inosine solution concentrations (M)
3.00x10-5 4.80x10-5
2.50x10-5 4.00x10-5
2.00x10-5 3.20x10-5
1.50x10-5 2.40x10-5
1.00x10-5 1.60x10-5
0.50x10-5 0.80x10-5
RANDOM RANDOM

The RANDOM concentration was 0.25*10^-5 for adenosine and 0.4*10^-5 for inosine.

Preparation of Dilutions of Adenosine


Preparation of Dilutions of Inosine


The groups then exchanged unknowns to determine the concentration from the calibration curves. In a week, the data will be revisited and the error will be propagated from the calibration curve to the concentration calculation. After making the calculation, the calculation of the unknown from the group will be compared.

Data

Adenosine Absorbance Spectrum at 260 nm


Adenosine Calibration Curve at 260 nm


Class Calibration Curve for Adenosine at 259 nm


Notes

Due to the time restrictions of the lab, the inosine spectra was taken the next lab (9/4/13).

Grubb's test was conducted to determine the outliers in the pooled class data.


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