Determining Concentration using a Spectrophotometer

by John Olson, Arlington High School, St. Paul, MN

Spectronic 20D+ and Vernier Spectrophotometer Program

Use of a colorimeter is described in the Extensions section at the bottom.

Background
One of the primary uses of a spectrophotometer is to determine the concentration of a solution.
The spectrophotometer shines light at a set color (wavelength) through a sample and the the
machine determines the percent that the light is absorbed by the sample. By comparing this
amount to a graph of known concentrations, the concentration of an unknown solution can be
determined.

The process that is usually followed is:

A. Determine the color of light that is absorbed the most by the solution. Often this wavelength
is given. You change the color (wavelength) of light shining through a concentrated sample of
the solution material and graph the absorbance.

B. Make known concentrations of the substance to be tested. For example: 20%, 40% etc. Set
the spectrophotometer to the wavelength determined in step A and measure the absorbance of
each known solution and graph absorbance vs concentration.

C. Measure the absorbance of the unknown and determine the concentration from the graph in
step B.

For practice experimentation you can use food coloring solutions or other colored solutions such
as .1M CuSO4 or .1M NiSO4 .

Procedure

General Setup
1. Turn on the Spectrophotometer and allow to stabilize for 15 minutes

2. Connect the interface cable to the modem port and start the computer.

3. Start the Spectrophotometer program

A. Determining the wavelength of maximum Absorbance (Absorbance vs. Wavelength).

1. Prepare a curvette (small test tube) with

2. Choose the wavelength portion of the program either from the button at the opening screen or
Wavelength under the Experiment menu.

3. With no sample in the spectrophotometer well, adjust the zero control (left knob) to read 0%
transmittance (mode set at Transmittance)

4. Follow the instructions on the screen to collect data

a. Set the desired wavelength with the knob on top. Press <return>.

b. Insert the blank (curvette with distilled water) and use the right control knob ot set 100.0%
transmittance. Press <return>.

c. Insert the sample into the well. When the transmittance reading is stable, click on the Keep
button or press <return>.

Repeat for each sample. You may wish to collect data for 50 nm intervals of wavelength. You
can collect closer interval close to the peak of the graph. When you are done collecting data,
click on the Stop button.

5. Try features of the Graph menu to enhance the graph, especially Big Dots and Connecting
Lines. Regression and statistics do not apply.

6. Determine the wavelength of the peak of the graph to use for future experiments. You can
also print the graph and save the data.

B. Making a calibration graph of Absorbance vs Concentration.

1. Set up 10 mL samples of varying concentrations. Make a known concentration such as 1

molar or .1 molar. Make dilutions of 20%, 40% 60% 80%, 100%.

2. Choose the Absorbance vs. Concentration button on the opening screen or choose
Concentration from the Experiment menu.

3. You will be prompted to remove samples from the well and use the zero control knob (left
side) to set 0.0% Transmittance. Click on the Okay button.

4. Set the wavelength (knob on top) to the wavelength from section A.. Insert the blank
(curvette with distilled water) and adjust the right transmittance control knob to 100.0%
Transmittance. Click on the Okay button.
5. Insert a sample and click on the click on the Keep button. Type the concentration in the C=
box and press <return>. Repeat this step with as many many samples as you want. ( You do not
have to recalibrate between samples. When you are finished collecting data, click on the Stop
button.

6. Enhance your graph using features from the Graph Menu. Make a Regression Line. You can
also display Statistics to get the equation of the regression line

7. Save or Print this graph for use with part C

C. Determining the Concentration of the unknown

1. Keep the settings the same from part B and have the graph of Absorbance vs Concentration
on the screen. . Select Interpolate from the Graph menu. Place the unknown solution into the
well. The computer will display the concentration of the unknown

Extensions

1. Use a Vernier Colorimeter with a Serial Box Interface or Universal Lab Interface(ULI) with
the Spectrophotometer or Logger Pro Software. The Colorimeter can also be use with the
Computer Based Laboratory (CBL) and graphing calculators. You cannot do part A -
Absorbance vs Wavelength since the colorimeter only produces three wavelengths. Choose the
wavelength that is most absorbed by the sample and use that for part B. Modify part B & C
based on the equipment and program

2. Water Testing: La Motte has test kits and standard solutions for testing water quality
indicators such as phosphate or nitrate.

John Olson - Arlington High School, St. Paul, MN, jolson@mail.ahs.stpaul.k12.mn.us

http://www.lcse.umn.edu/~jolson/jolson.html