SPECTROSCOPYSPECTROSCOPY

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.

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.

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.

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

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

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

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

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.

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.

Kendall Olds

Derrick Woods

Ricketta Johnson

Carl Flowers