1. LAB 3 The Spectrophotometer: Measuring Concentration Using Absorbance

2. Measuring Absorbance using a Spec • set-up: • molar mass of KMnO4 = 158.03g • 0.1mg/ml KMnO4 solution = 0.63mM

3. Determining Concentration from Absorbance – The Standard Curve • set-up: • multiple test tubes prepared with decreasing concentrations of KMnO4 • prepared a two-fold serial dilution • prepared more serial dilutions – prepared 7 tubes • test tube #1 = 0.1 mg/ml • test tube #7 = 0.0015625 mg/ml • each tube read at Abs 545nm 2-fold dilution 2.5 ml 2.5 ml 2.5 ml 2.5 ml 2.5 ml 5.0 ml sample 0.1mg/ml 64-fold dilution 2.5 ml water for dilution

4. Unknown • unknowns read at Abs 545nm • three unknowns of various colors/concentrations • EXPERIMENTAL APPROACH: measure the absorbance of these three unknowns and compare them to your standards • requires determining the molar absorptivity • this is calculated using your standards and the slope of a Beer-Lambert plot

5. KMnO4 Standard Curve

6. Beer’s Law • Beer’s Law relates the absorbance of light to the properties of the material through which it is passing • A = ebc • A = absorbance of the sample at a given wavelength • e = molar absorptivity of the sample (no units) • b = sample path length of the sample (cm) • c = concentration of the sample (same units as your standard) • rewriting the equation gives you • c = A/eb • using a spectrophotometer you can determine A for any unknown • you know the path length of the sample = 1 cm wide test tube • so b= 1 • PROBLEM: unknowns in this equation are e and c

7. Molar absorptivity • molar absorptivity (e) can be obtained by determining the slope of a standard curve • e is the coefficient in the equation y = mx • so knowing e means you can now calculate c cunknown = Aunknown/eb -b is 1cm -units are whatever the units are for the standards

8. KMnO4 Standard Curve and Unknown slope = 0.386 – 0.088 0.1-0.025 slope = 3.973

9. How’d we do? • 0.1 mg/ml KMnO4 is 0.63 mM • 158.03 mg in 1 ml of water is 1MKMnO4 • one of my unknowns was 0.051 mg/ml = 0.323 mM • if 0.1 mg/ml is 0.63 mM • then 0.051 mg/ml is 0.32 mM • the other unknowns • 0.273 mg/ml = 1.72 mM • 0.151 mg/ml = 0.951 mM