SPECTROSCOPY
Kendall Olds
 Derrick Woods
 Ricketta Johnson
 Carl Flowers

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.
Spectrophotometry
What is it????

The process of measuring light intensities with a
spectrophotometer.
Spectrophotometer
What 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.
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.
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.
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.
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.
Standard Curve for Methylene Blue
Demonstration From Dr. Uddin
Results From Students
Concentrations %
Concentrations % Absorption
0.0001
0.001
0.01
0.1
1
unknown
Absorption
0.015
0.011
0.029
0.183
0.454
0.164
0.0001
0.001
0.01
0.1
1
unknown
0.01
0.024
0.026
0.231
0.516
0.13
Regression Analysis
from Dr. Uddin’s Experiment
Regression Statistics
Multiple R
0.958350388
R Square
0.918435465
Adjusted R Square
0.891247287
Standard Error
0.144047944
Observations
5
R Square is closed to 1, so it is a
good of fit for a linear regression
Intercept
Absorption
Coefficients
-0.082
2.199
Concentrations %
Absorption Line Fit Plot
1.5
Concentrations
%
1
0.5
0
-0.5 0
0.2
0.4
0.6
Predicted
Concentrations
%
Absorption
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 Students’ Experiment
Regression Statistics
Multiple R
0.9437
R Square
0.8906
Adjusted R Square
0.8542
Standard Error
0.1668
Observations
5
R Square is closed to 1, so it is a
good of fit for a linear regression
Intercept
Absorption
Coefficients
-0.082
1.888
Concentrations %
Absorption Line Fit Plot
1.2
1
0.8
0.6
0.4
0.2
0
-0.2 0
Concentrations
%
0.2
0.4
0.6
Predicted
Concentrations
%
Absorption
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.
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
Spectrophotometric Determination &
Protein Concentrations
Intercept
Absorption
Concentrations(mg/ml)
It is good of fit.
Absorption Line Fit Plot
Protein
Regression Statistics
Multiple R
0.9780
R Square
0.9565
Adjusted R Square
0.9503
Standard Error
0.0654
Observations
9
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.000 1.000 2.000 3.000
Protein
Concentrations(
mg/ml)
Predicted
Protein
Concentrations(
mg/ml)
Coefficients
Absorption
-0.808 The regression line is
0.977 % 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.
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.
This Concludes Our Presentation
We thank TLSAMP, Dr. Uddin, and Dr. Chu for helping and
supporting us with this project.

Derrick Woods


Kendall Olds
Ricketta Johnson

Carl Flowers