1. Determination of Concentration Using Spectrophotometry
Lab 8

2. Outline Purpose Qualitative Distinction Spectrophotometry Beer’s Law
Calibration Curve
Procedure
Points of Interest
Waste
Important - printouts
Next Lab Reminder

3. 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.

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 could be due to Cu2+ (it is in fact food coloring, not Cu2+).
The color of cranberry juice is due to anthocyanins.

5. Qualitative Distinction
The color we see is the color of light transmitted, or “getting through” the solution. We see the color of light “left over” after some wavelengths have been absorbed.
Today we will see blue because the solution absorbs orange wavelengths and what’s left over appears blue.

6. Qualitative Distinction
The intensity of the 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 the 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. 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 absorb!

11. 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.

12. 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.
Using the calibration curve equation, the concentration of the unknown solution can be calculated, given the %T or absorbance of the unknown solution:
Unk Abs = m [unk cs] + b, therefore, [unk cs] =

13. Points of Interest MicroLAB™ colorimeter operation Cuvet handling
Your instructor will run through the basics.
Cuvet handling
Wipe down the cuvet with a KimWipe before insertion into the interface.

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

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

16. Important: Before you leave the lab today, print off the following:
Colorimeter spreadsheet
Unknown Abs vs. [ ] graph
Spectrum Profile #1

17. Lab 9 Reminder
Lab 9 next week.