Determining the Concentration of a Solution Using Absorbance
Spectroscopy (Beer’s Law)
Purpose: To construct a standard curve of absorbance vs. concentration for 5 solutions of known
concentration; use your standard curve to determine the molar concentration of an unknown
solution.
Introduction
As discussed earlier in the course, most chemical reactions in the laboratory take place in
solution. In nature, “pure” water does not exist; the water always contains substances dissolved
in it. Chemists test our drinking water to make sure that the concentrations of different
substances, especially very toxic heavy metals, are at an extremely low concentration. One
important technique for quantitatively measuring the exact concentration of solute present is to
measure the light absorbance of the solution: The higher the light absorbance, the higher the
concentration. This relationship is called Beer’s Law after its discoverer and can be expressed
as:
A = εcl where
A = absorbance,
ε = extinction coefficient (a measure of how strongly a particular substance absorbs light at a
given wavelength wavelength
c = the concentration measured in moles/liter (M)
l = the pathlength (the distance the light has to pass through the test tube holding the solution.)
Note in most experiments, the pathlength is 1 cm, and since the value is 1 it can be ignored in the equation.
Concept of Beer’s Law: The number of photons of light (intensity) of light is entering and
exiting a solution are measured and compared.
2 / 5 photons absorbed
3 / 5 photons absorbed
In the figure at left: 5 photons of enter the test tube; 3 photons are detected as having
passed through; thus 2 photons out of 5 were absorbed. Concentration (solute
particles per volume) is directly to the proportional number of photons of absorbed.
Background
When atoms absorb light energy, electrons make transitions or quantum leaps from lower energy
levels to higher energy levels. The energy absorbed in making the quantum leap is specific to the
atom. The energy of the transition is quantized and is specific and unique for each individual
atom or atom that is part of a compound or molecule.
Concept electron absorbing
energy within atom
Many possible jumps are present within atoms with many electrons, producing a more complex
absorbance graph. Note that although copper sulfate looks blue, it actually strongly absorbs red.
Red, blue light
Red light
absorbed
Choosing an appropriate wavelength of light
Recall that the wavelength of light is directly linked to the energy of the light. In order to
measure a large absorbance in experiment, we need to choose a wavelength For example, in the
absorbance vs wavelength graph for a solution of copper sulfate notice that solutions absorbs
strongly from approximately 600 to 800 nm, with the maximum absorbance is around 750 nm.
Thus in an experiment we would chose a wavelength within that range. Note that there is an
important distinction between the color of the light we observed reflected and the color the light
absorbed. A wavelength of 750 nm corresponds to red light, while a solution of copper sulfate
appears blue. Copper sulfate reflects or transmits blue light, but absorbs red light.
Light that appears red to us is at a wavelength between 630 and 700 nm. An
object that appears red is reflecting light at a wavelength in this range and is absorbing all
other wavelengths of visible light. Blue light shining on a blue solution would simple be
absorbed, causing the solution to appear colorless.
Blue transmitted
Pre-Lab Questions
1. a. If you are wearing a red t-shirt, what color(s) of light is/are it reflecting?
b. What color(s) is/are absorbed?
2. If a compound has the spectrum below what color is it?
Safety
Wear goggles.
Materials
Beaker, 250 mL
Pipettes, 6
Cuvettes, 7
Spectrophotometer
Tissues or lens paper
Wash Bottle
Data Table
Concentration of
Stock Solution
Blank
0M
1M
2M
3M
4M
6M
Color Comparison
(Rank Solutions*)
0
%T (measured from
spectrophotometer)
Absorbance
(calculated from %T)
* Rank Solutions from lightest blue = 1 to darkest blue = 5, mark the blank as 0.
Unknown
______
______
Procedure Part 1: Preparation of the Lab
1. Turn on the spectrophotometer. The spectrophotometer is the instrument used to
measure light absorbance. It will take at least 15 minutes to warm up so look at the
clock and record the time. Do the rest of the steps in Part I while you wait for it to
warm up.
2. Turn the wavelength knob on the spectrophotometer to 635 nm.
3. Get distilled water (in wash bottle) and a 250 mL beaker (for waste).
4. Take out a sheet of paper and label it with each of the stock solution molarities.
5. Take one of the cuvette to the stock solutions, and using the correct pipet, fill the
cuvette about ¾ full with one of the solutions. Go back to your seat, wipe the outside
of the cuvette with a tissue to clean it and place that cuvette on your sheet of paper
(match the molarity of the solution to the labeled location on your paper).
6. Repeat step 5 a total of 4 more times until you have one of cuvette of each of the
stock solutions.
7. Compare the colors of each of the solutions. Rank them on a scale of 1-5 with 1
being the lightest and 5 being the darkest. Fill in the second row of the data table.
8. Fill one of the remaining 2 cuvets about ¾ full with distilled water from your wash
bottle. Wipe the outside of the cuvet with a tissue to clean it.
9. Look at the clock and see if 15 minutes have passed. If so, go on to Part II. If not,
wait until it’s been 15 minutes.
Procedure Part II: Testing the Stock Solutions
10. Pick up the cuvette that is full with distilled water. Wipe the cuvette with a tissue,
and then place the cuvette in the spectrophotometer.
11. Press the 0 Abs/100%T button on the spectrophotometer to set 0 Absorbance.
12. Remove the “blank” (distilled water) cuvette from the spectrophotometer
compartment.
13. Pick up the cuvette with the first stock solutions. Wipe the cuvette with a tissue, and
then place the cuvette in the spectrophotometer.
14. Record the absorbance reading in the Data Table.
15. Remove the stock solution cuvette from the spectrophotometer. Set 0 Absorbance
with the blank cuvette as done in steps 10 and 11. Remove the blank and replace it
with the second stock solution cuvette as done in step 13.
16. Record the absorbance reading in the Data Table.
17. Repeat steps 15 and 16 with the remaining stock solutions. Record all absorbance
readings in the Data Table.
18. Using the values you obtained from the spectrophotometer, Plot a calibration curve
for question 1 in the post-lab.
Procedure Part III: Testing the Unknown Solution
19. Take the last cuvette and fill it ¾ full with the unknown solution. Wipe it clean and
set it down on your table next to the stock solutions.
20. Prediction. Answer Post Lab Question 2.
21. Repeat steps 10-12 to re-zero the spectrophotometer.
22. Repeat step 13 with the unknown solution.
23. Record the absorbance reading in the Data Table.
24. Check that the unknown absorbance makes sense by comparing it to your prediction
from step 20.
25. If your values all seem to make sense (check the graph and your unknown
absorbance), then you may clean up following your teacher’s instructions.
Post-Lab Questions
1. Plot a calibration curve from the absorbance values of the stock solutions obtained from
the spectrophotometer. Remember: Absorbance on the y-axis versus Concentration on
the x-axis.
2. Prediction: By comparing the color of the unknown solution to the stock solutions,
predict the concentration of the unknown. (Hint: Which two stock solutions does the
color fall between? Does it match one of the stock solutions?)
3. Compare the concentration of each stock solution to the color ranking in your Data Table.
What is the relationship between the concentration of a solution and its color intensity?
4. Compare the concentration of each solution with its absorbance. What is the relationship
between concentration and absorbance?
5. Now, look at the calibration curve that you made in question 1. Would it make sense that
the relationship between concentration and absorbance should include the origin (0, 0) as
a point? Explain your reasoning.
6. Using your answer to Question #5, draw the “best-fit” straight line through the data
points.
7. Use your graph of Absorbance vs. Concentration to estimate the concentration of the
unknown solution.