Lab Worksheet: Spectrophotometry
Learning Outcomes - Upon completion of this worksheet, students will be able to do the following:
calculate concentrations in a serial dilution
plot absorbance vs. concentration data in Excel
remove non-linear data
extract the extinction coefficient from a standard curve
use a standard curve trendline to calculate a concentration based on an absorbance
Part 1: Making a standard curve
You're given a stock solution that contains 0.018 mg/mL or potassium ferricyanide and instructed to perform a 2-fold serial dilution and make a standard curve of the absorbance at 420 nm. The first entry in the table below is the undiluted stock. Fill in the table by calculating the concentrations in the dilution. Then plot the data in Excel.
|
Concentration (mg/mL)
|
Abs @ 420 nm (AU)
|
|
0.018
|
2.5
|
|
|
2.6
|
|
|
1.8
|
|
|
0.95
|
|
|
0.45
|
|
|
0.22
|
|
|
0.1
|
|
|
0.002
|
|
|
-0.05
|
Sketch the plot with all data included to the right of the table above. Include a title, labeled axes with units, and the linear trendline equation with the value for R2.
Remove any data points that deviate from the linear response predicted by Beer's Law, replot your data, and sketch the plot below with all the same details as above (title, etc.)
Follow-up questions:
1. What is the trendline equation for the standard curve above?
3. What is the extinction coefficient with units?
4. If you analyze a sample of ferricyanide with an unknown concentration, and it has an absorbance of 0.30 AU at 420 nm, what is the concentration of that unknown?
5. What are two pieces of glassware or measurement tools that are NOT appropriate (with respect to accuracy and precision) for making serial dilutions?