After setting the spectrophotometer to 0 absorbance (100% transmittance) at 580 nm with a cuvette containing water (blank), measure the absorption of the samples in sample tubes #1-#5 at 580 nm.
1. Set the Spec-20 (spectrophotometer) to a wavelength of 580 nanometers.
2. Zero (Blank) the Spec-20 using water as the blank.
3. Read the absorbance of your solutions from the lowest concentration to the highest concentration. Pour the solution from tube #5 into a clean, dry cuvette. Wipe any fingerprints from the outside of the cuvette with a Kimwipe. Place the cuvette into the Spec-20, making sure the clear side of the cuvette faces you.
4. Dispose of the solution.
5. Rinse the cuvette with the next standard and dispose of the rinse.
6. Repeat steps 3-5 for the remaining four diluted standard solutions. Determine the absorbance of test tube #4, then #3, then #2, and lastly #1.
7. Obtain two solutions of BPB of unknown concentration from your instructor.
Procedure Part B: Graphing the Calibration/Standard Curve
Plot the absorbance (y-axis) versus concentration of the BPB (x-axis). Draw a best-fit line using your data points. Do this by hand and with a computer. Determine the slope of the lines for each graph. You now have a working relationship between the absorbance and the concentration of BPB.
Data Table 2: Absorbance and Concentration of Unknown Solutions
Unknown #
Absorbance
Concentration (ppm)
1
0.220
2
0.632
1. Calculate the ppm of each sample using:
a) The slope of your hand-drawn graph
b) The slope of your computer-drawn graph
c) The Beer-Lambert Law using the molar absorptivity coefficient determined in part A
2. Does the calibration curve obey the Beer-Lambert Law? Why?
3. What is the percent error for the hand-drawn slope compared to the computer-generated slope (actual)?
4. If you rinsed each solution with water between each reading and neglected to dry it completely, how would that affect your results.