Measurement of concentration in coloured solution by absorption
spectrophotometry
Theoretical introduction
Absorption spectrophotometry is a method for determining the concentration of the dispersed
fraction in an analytical dispersion, based on the measurement of absorption of light. When
monochromatic light passes through the dispersion, the light intensity decreases from the original
value to the value . This decrease in intensity is expressed as the absorbance (the equivalent
term extinction is also sometimes used). The absorbance is defined as
( )
Another quantity expressing the absorption of light is the transmittance , defined as
The absorbance and transmittance are therefore related through the formula
these quantities are dimensionless.
. Both of
The absorbance depends on the concentration c of the dispersed fraction (i.e. solute in a solution)
linearly, through the Lambert-Beer Law:
where is the extinction coefficient, the value of which is specific for the given solute and for the
given wavelength of the light (the unit of
is
), is the molar concentration of the
solute (mol/l), and is the thickness of the sample with the solution (cm).
Absorption photometry can be used to determine the concentration of both colorless and colored
analytical dispersions. For colorless solutions, monochromatic light from the UV range (200-380 nm)
is used. For colored solutions, either UV light or light from the visible range of the spectrum (380-760
nm) may be used. One should select the specific wavelength that permits the highest sensitivity of
the measurement procedure. This means finding the wavelength for which a small change in
concentration leads to the largest change in absorbance. The wavelength satisfying this requirement
is the wavelength of the peak of the absorption spectrum; when multiple absorption peaks are
present in the spectrum, one chooses the wavelength of the peak with highest absorbance.
In this excercise, you will use a modern type of spectrophotometer (Biochrom Libra S6), which
permits a quick measurement of the absorption spectrum across a wide range of wavelengths. A
halogen lamp with tungsten filament is used as the light source; this lamp emits light in the whole
visible light spectrum. The white light is collimated, passes through the sample with the measured
solution, and then is diffracted on an optical grating, resulting in separation into monochromatic
beams. The beams are incident on an array of photodiodes, which measure the intensity of the
individual beams. The apparatus therefore does not contain any moving parts, and permits a
simultaneous measurement of the absorbance values for up to 65 distinct wavelengths.
Equipment:
1.
2.
3.
4.
Absorption spectrophotometer Biochrom Libra S6.
Computer with interface for communication with spectrophotometer.
Set of CuSO4 solutions.
Plastic cells to hold solutions.
Task:
1. Measure the absorption spectrum of copper sulfate in solution.
2. Verify the validity of the Lambert-Beer law.
3. Determine the unknown concentration of copper sulfate in the provided unmarked solution.
Execution:
1. Switch on the the spectrophotometer, using the black key (button) in the lower right corner
of the keypad. Make sure that no sample is inserted in the cell holder. Wait for 1 minute until
the self-diagnostic test is completed. (Whenever the spectrophotometer is busy with
diagnostics or measurement, the line at the very bottom of the display will keep changing).
2. By pushing the F2 key, access the home screen, which gives the following choices:
• Repeat last operation
• Make a measurement
• Set up instrument
You can return to this screen at any later time by repeatedly pushing the red key.
3. Switch on the computer. Start the Grafico program (use the icon in top right corner of the
computer screen). Fill in your name (one student is enough), and give the number of your
group as the Organization. Once you reach the main window, switch Grafico to Scan mode
using the 5th button on the window’s toolbar. An empty graph should appear.
4. Go back to the spectrophotometer. Select „Make a measurement“ and at the next screen,
select „Scan“. Then use the F1 key to select the absorbance (Abs) as output.
5. Insert plastic cell filled with pure water into the cell holder located to the right of the screen.
This will be your reference sample. Note: all cells should be inserted with the same
orientation, such that the arrow mark near top of the cell faces the arrow on top of the
holder.
6. Push the blue key marked „0A/100%T“ to start the measurement of the absorption
spectrum of the reference sample; it will be automatically saved in the spectrophotometer
memory.
7. Take out the reference sample and insert the cell filled with copper sulfate solution (use the
highest concentration available). Then, push the green key to start the measurement of the
absorption spectrum. On the display, a graph will appear, with the wavelength on the x-axis
and the difference of absorbances of the reference sample and the current sample on the yaxis. Does the absorption spectrum you measured agree with the color of the solution as
perceived by your eyes?
8. The graph will appear as well on the computer, in the Grafico window. Save the graph to
Desktop as a pdf file (use the Print command, not the Save command). Then save the file to
your USB drive or send by e-mail.
9. From the graph, read off the wavelength of the absorption peak. All subsequent
measurements should be done at this wavelength.
10. Switch the spectrophotometer to the single wavelength mode (Home screen → Make a
measurement → Single/Multi/Ratio → Single λ). Set the wavelength to the value you found
in the previous step (select Set λ, then use the arrow keys to modify the wavelength, then
select Accept λ).
11. One by one, insert cells filled with solutions of known concentrations, and then with the
unknown concentration. For each solution, use the green key to measure the value of the
absorbance (relative to the absorbance of the reference sample, which is still saved in the
memory).
12. Make a table of the recorded absorbance values. Construct the calibration curve (the graph
of absorbance as function of the concentration of solution). Does the curve agree with the
Lambert-Beer law? Use the calibration curve to read off the concentration of copper sulfate
in the solution with unknown concentration.
13. Clean the used cells. Switch off the spectrophotometer. Delete the file you created from the
computer. Exit the Grafico program.
Questions:
1. Which types of solutions can be analyzed using the Libra S6 spectrophotometer to determine
their concentration?
2. What is the definition of absorbance (extinction) and transmittance?
3. What does the Lambert-Beer law say?
4. What is the absorption spectrum?