1. Determination of Concentration Using Spectrophotometry
Lab 8

2. Purpose
This experiment demonstrates the linear relationship between the absorbance and the concentration of a colored solution.
Beer’s Law will be used to determine the concentration of a sample for which the concentration is unknown.
A relationship can be observed between color intensity and the concentration of a solution.

3. What is Absorption?
Absorption occurs when a photon is targeted at a substance and causes an electron in the substance to go from a lower energy level to a higher energy level.
Absorbance is a mathematical expression of the amount of absorption that occurs.

4. Qualitative Distinction
Chemical solutions owe their color to light-absorbing species in the solution, whether these are ions or complex molecules. For example:
The blue color of today’s solution is similar to that of a Cu2+ solution. (You will be using food coloring in this lab, which does not contain Cu2+.)
The color of cranberry juice is due to anthocyanins.
Anthocyanins
FD&C Blue #1

5. Qualitative Distinction
The color we see is the color of light transmitted or “passed through” the solution. We see the color of light “left over” after all the other wavelengths have been absorbed.
In this lab we will see blue because the solution absorbs all other wavelengths except blue. All the other wavelengths combined gives us the color orange, the complimentary color to blue.

6. Qualitative Distinction
The intensity of the absorbed color is proportional to the concentration of the absorbing chemical species.
We can do a qualitative distinction by eye, or
We can do a quantitative measurement by spectrophotometry.

7. Spectrophotometry Spectrophotometers shine a light through a sample.
Some detect light from only one wavelength and some detect light from all visible wavelengths. The MicroLAB™ spectrophotometer emits and detects light from sixteen different wavelengths.
The light that is not absorbed by the sample, but transmitted instead, hits a light detector.
The spectrophotometer calculates the percentage of light transmitted.
It then uses an algorithm (formula) to convert percent transmittance to absorbance.

8. Spectrophotometry Transmitted Light Incident Light I0 I Light Source
Wavelength Selector
Sample
Detector b
b = mm or cm

9. Spectrophotometry
Transmittance: %T = x 100%
Absorbance: Abs = log

10. Factors affecting Absorbance
Inherent
Potential Interference
Concentration of the analyte
Matrix effects of the solvent
Pathlength
Dirty or scratched glassware (cuvets / vials)
Molar absorptivity constant (determined by the nature of the analyte and wavelength of light measured)
pH of the solution
Presence of interfering substances (chemical)
Transmittance
Temperature

11. Beer’s Law Abs = ε b C where Abs = absorbance (no units)
ε = molar absorptivity (M-1cm-1)
b = path length (cm)
C = concentration (M)
These measurements all take place at the wavelength at which our absorbing species absorbs light.
By plotting Beer’s Law on a graph, we can establish a calibration curve with our standard solutions.

12. Calibration Curve Plotting Abs vs. [colored solution] gives:
This is called a “calibration curve.”
For y = m x b
Abs = m [colored solution] + b
Abs = ε b c
After the calibration curve is established with your standard solutions, the equation is used to calculate the concentration of the unknown solution, given the absorbance of the unknown solution.
Important: The unknown MUST contain the same analyte as the solution used to make the calibration curve.

13. Procedure To obtain the calibration curve:
Prepare a series of colored solutions of known concentration (“standards”).
The absorbance of each solution is measured.
Absorbance versus concentration is plotted.
To find the unknown concentration:
Using the calibration curve equation and the absorbance of the unknown solution, the concentration of the unknown solution can be calculated:
Unk Abs = m [unknown colored solution] + b
[unknown colored solution] =

14. Points of Interest MicroLAB™ LED spectrophotometer operation
Your instructor will run through the basics.
Cuvet handling
Wipe down the cuvet with a KimWipe before insertion into the interface.
Blank Solution
The blank solution is used to correct for the matrix effects of the solvent.

15. Safety Concerns – Food Dye
Avoid contact with skin and eyes.
Do not inhale vapor or spray.
Do not ingest.

16. Waste
All waste solutions can be disposed of down the sink, with plenty of water.

17. Important: Before you leave the lab today, print off the following:
Spreadsheet
Unknown Abs vs. [ ] graph
Spectrum Profile of solution #1

18. Next Week – No Labs
Happy Spring Break!