1. Absorbance spectroscopy
A tutorial
by Dr. Lara Baxley
Cuesta College
San Luis Obispo

2. Note
This tutorial may not work well on a mobile device. If you are using a mobile device, some images and equations may not display well.
In that case, you are advised to use a computer with power point.

3. Introduction to this tutorial
This is a self-paced tutorial about absorbance spectroscopy.
Click your mouse or down arrow key to reveal each part.
If the tutorial asks a question, you should answer it either in your head or write down your answer before advancing (have a calculator handy).
Take notes on this material; you will need it to complete your prelab and lab calculations.

4. What makes a solution appear a certain color?
A solution will appear a certain color if it absorbs the complementary color (the opposite color in the color wheel).

5. The Color Wheel
For example: If a solution appears red, this means that it is primarily absorbing green.
Sample absorbs green, but transmits all other colors
White light containing all colors shines on the sample
Eye sees the remaining combination of colors as red

6. A Spectrophotometer
A spectrophotometer measures the amount of light absorbed by a sample.
Here’s how it works:
3. A diffraction grating separates the colors of light
5. Light passes through the sample
1. A light bulb emits white light
2. Light passes through a slit to form a narrow beam
4. Another slit allows just one color to pass
6. A detector measures the final amount of light

7. Concept Check:
Why would a solution of the food coloring dye Blue #2 look blue?
The solution of the blue dye strongly absorbs orange light at about 600 nm.
The solution of the blue dye  strongly absorbs blue light at about 450 nm.
The answer is (a). If the solution absorbed blue light, it wouldn't be reflected or transmitted for us to see.

8. Absorbance
Absorbance, A, is a measure of how much light is absorbed. Absorbance does not have units.
The less light that gets through, the greater the absorbance.

9. Absorbance spectrum
The absorbance spectrum is a graph of the absorbance of a solution at different wavelengths. An absorbance spectrum might look something like this:
Notice that there is a peak at 600 nm. What color would you expect this solution to be?
Since the solution is absorbing orange, it must appear blue, which is complementary to orange.

10. lmax The wavelength of maximum absorbance is called lmax.
For best accuracy, when measuring the absorbance of several solutions, it is best to measure as close to lmax as possible.

11. A Colorimeter
The Vernier Colorimeters used at Cuesta College are different from spectrophotometers.
A colorimeter uses LED lights that emit specific wavelengths.
This means that a colorimeter can only measure absorbance at these specific wavelengths.
With a colorimeter, the wavelength closest to lmax is usually selected.
635 nm
565 nm
470 nm
430 nm

12. Using a Colorimeter The sample goes in the sample holder.
A colorimeter contains the LEDs, a sample holder, and a detector.
The sample goes in the sample holder.
Select the best wavelength for an orange solution.
For an orange solution, the best wavelength is 470 nm.
635 nm
565 nm
470 nm
430 nm
LEDs
detector
sample
holder

13. Using a Colorimeter
Question: What would happen if the wrong wavelength were selected?
If the wrong wavelength is selected there will be little to no absorbance.
All four wavelengths could be tested (one at a time) to determine the maximum absorbance.
635 nm
565 nm
470 nm
430 nm
LEDs
detector
sample
holder

14. Concentration and Absorbance
Which of these two solutions contains a higher concentration of red dye?
Did you answer solution A? If so, you are correct! A higher concentration leads to a darker color.

15. Concentration and Absorbance
Which of these two solutions will have a higher absorbance at lmax?
Did you answer solution A again?
That’s right! The higher the concentration, the greater the absorbance.

16. The Beer-Lambert Law A = ebc
The mathematical relationship between concentration and absorbance is called Beer-Lambert Law. It looks like this:
A = ebc
b = 1.00 cm
The parts of the equation are:
A = absorbance
= molar absorptivity (constant for a given solute at a given wavelength)
b = width of the tube holding the sample (1.00 cm in our lab)
c = molar concentration (mol/L)

17. Concept Check: the Beer-Lambert Law: A = ebc
In the above equation, which variable(s) can change for a specific substance at a fixed wavelength?
Concentration
The molar absorptivity (e)
Absorbance
The answers are (a) and (c). If concentration increases, so would absorbance. The value of e doesn’t change.

18. Using Absorbance for a Calibration Graph
Is it possible to calculate the concentration of a colored solution after measuring its absorbance in a colorimeter?
Yes!
A scientist first makes a calibration graph using absorbance values from solutions with known concentrations.
The concentration of the unknown solution is then calculated using the calibration graph and the solution’s absorbance.

19. Using Absorbance for a Calibration Graph
Imagine that you measured the absorbance of these 5 solutions of known concentration:
What trend do you predict for their relative absorbance readings at lmax?

20. Using Absorbance for a Calibration Graph
The actual absorbance readings are shown here:
A = ebc
Look at this data and look at the Beer-Lambert law.
What variables would you graph in order to make the data fit a straight line (y = mx + b)?

21. Making a Calibration Graph
A = ebc c
y = m x + b
If you were to graph this data, what variable would you graph on the y-axis?
Absorbance, because it’s the dependent variable (it’s also on the left of the equals sign).
What would you graph on the x-axis?
Concentration, because it’s the independent variable (it’s also on the right of the equals sign).
Let’s see how this works…

22. Making a Calibration Graph
A = ebc c
y = m x + b
(mol/L)

23. Using a Calibration Graph
A = ebc
Here is an actual graph of this data. This is also called a calibration graph because it is made using known values and can be used to determine an unknown. Example: If an unknown solution has an absorbance of 0.351, what is its concentration? (calculate this before you click!) = 4.806x – x = c = M

24. Concept Check:
Once the absorbance of an unknown solution is measured, how is its concentration determined?
Use the absorbance of a single calibration solution to calculate the molar absorptivity.
The absorbance of the unknown is used in the equation of the best fit line from the calibration graph.
Use the Beer-Lambert law and the molar absorptivity from a single calibration solution.
The answer is (b). Using the equation from the graph is more accurate than a single data point.

25. Determining e from the Calibration Graph
A = ebc
y = m x + b
Using the graph, calculate the molar absorptivity of this substance, including units. Hint: Remember that b = 1.00 cm Looking at the equation above, m = eb, therefore,
e = m b =
4.806 M-1
1.00 cm
= M-1cm-1

26. Determining e from the Calibration Graph
A = ebc
y = m x + b
How do you figure out the units of the slope? Slope = rise/run or Dy/Dx Therefore, the units of the slope are the (y-axis units)/(x-axis units) Remember that A does not have units.

27. Conclusion
Now that you have completed this tutorial you should be able to,
Predict the relationship between a solution’s color and the wavelength of light it absorbs.
Understand how spectrophotometers and colorimeters measure the absorbance of a solution.
Create a calibration graph and use it to determine the concentration of an unknown solution.

28. The End