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SPECTROSCOPYSPECTROSCOPY
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Kendall Olds Derrick Woods
Ricketta Johnson Carl Flowers
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Definition of Spectroscopy
The study of how the chemical compound interacts with different wavelengths in a given region of electromagnetic radiation is called spectrochemical analysis.
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Spectrophotometry What is it????
The process of measuring light intensities with a spectrophotometer.
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SpectrophotometerWhat is it????
A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the color, or more specifically, the wavelength of light.
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How to use a spectrophotometer
Turn the spectrophotometer on at least 10 minutes before using.
Set the wavelength desired using the knob (upper right-hand).
With the chamber empty and closed, adjust the machine to read 0% using the front left-hand knob.
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How to use a spectrophotometer
Insert a clean sample tube containing at least 3 ml of distilled water into the chamber. The outside of the sample tube must be wiped clean using a Kleenex tissue because fingerprints will be read by the machine.
Adjust the absorbance to 0 using the front right-hand knob.
Insert a clean sample tube containing the light absorbing sample (at least 3 ml.). Read the absorbance.
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The Procedure
1. First we made a series of solutions of known concentration of methylene blue.
2. We set the mode of the spec 20(spectrometer) to absorption and set the wave length to 668.
3. We then measured the absorption of the standard solution from low concentration to the highest concentration.
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The Procedure
4. Each time we took a reading we set the absorption to zero setting with the distilled water.
5. We then recorded the absorption of the unknown solution.
6. Then we constructed a standard curve using the data from the know solution.
7. From the standard curve we estimated the strength of the unknown solution.
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Concentrations % Absorption
0.0001 0.0150.001 0.0110.01 0.0290.1 0.183
1 0.454unknown 0.164
Standard Curve for Methylene Blue
Concentrations % Absorption
0.0001 0.010.001 0.0240.01 0.026
0.1 0.2311 0.516
unknown 0.13
Demonstration From Dr. Uddin Results From Students
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Absorption Line Fit Plot
-0.5
0
0.5
1
1.5
0 0.2 0.4 0.6
Absorption
Conce
ntr
atio
ns
%
Concentrations%
PredictedConcentrations%
Regression StatisticsMultiple R 0.958350388R Square 0.918435465Adjusted R Square 0.891247287Standard Error 0.144047944Observations 5
CoefficientsIntercept -0.082Absorption 2.199
R Square is closed to 1, so it is a good of fit for a linear regression
The regression line is
% of concentration = 2.199 * absorption – 0.082
When the absorption is 0.165, the unknown of concentration is 2.199 * 0.165 – 0.082, which is 0.2785.
Regression Analysis from Dr. Uddin’s Experiment
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R Square is closed to 1, so it is a good of fit for a linear regression
The regression line is
% of concentration = 1.888 * absorption – 0.082
When the absorption is 0.13, the unknown of concentration is 1.888 * 0.13 – 0.082, which is 0.1629.
Regression Analysis from Students’ Experiment
Absorption Line Fit Plot
-0.20
0.20.40.60.8
11.2
0 0.2 0.4 0.6
Absorption
Conce
ntr
atio
ns
%
Concentrations%
PredictedConcentrations%
CoefficientsIntercept -0.082Absorption 1.888
Regression StatisticsMultiple R 0.9437R Square 0.8906Adjusted R Square 0.8542Standard Error 0.1668Observations 5
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Spectrophotometric Determination & Protein Concentrations
Protein Concentrations(mg/ml)
Absorption
group I group II group III average
0.1 1.01 1.03 1.05 1.03
0.2 1.03 1.04 1.07 1.05
0.3 1.07 1.08 1.10 1.08
0.4 1.20 1.09 1.27 1.19
0.5 1.36 1.37 1.42 1.38
0.6 1.44 1.44 1.48 1.45
0.7 1.48 1.48 1.50 1.49
0.8 1.56 1.56 1.58 1.57
1.0 1.90 1.90 1.95 1.92
unknown 1.08 1.08 1.08
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Spectrophotometric Determination & Protein Concentrations
Regression StatisticsMultiple R 0.9780R Square 0.9565Adjusted R Square 0.9503Standard Error 0.0654Observations 9
Absorption Line Fit Plot
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0.000 1.000 2.000 3.000
Absorption
Pro
tein
Concentr
ations(m
g/m
l)
ProteinConcentrations(mg/ml)
PredictedProteinConcentrations(mg/ml)
It is good of fit.
CoefficientsIntercept -0.808Absorption 0.977
The regression line is% of concentration = 0.977 * absorption – 0.808
When the absorption is 1.08, the unknown of concentration is 0.977 * 1.08 – 0.808. which is 0.247.
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Conclusion
From this project, we learn the way to use a spectrophotometer and collect the data from the biology lab. After that we do the regression analysis from the statistics class. The results show well and the unknown concentrations have been found.
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This Concludes Our Presentation
We thank TLSAMP, Dr. Uddin, and Dr. Chu for helping and supporting us with this project.
Kendall Olds
Derrick Woods
Ricketta Johnson
Carl Flowers