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LAB 3. The Spectrophotometer: Measuring Concentration Using Absorbance. Measuring Absorbance using a Spec. set-up: molar mass of KMnO4 = 158.03g 0.1mg /ml KMnO4 solution = 0.63mM. Determining Concentration from Absorbance – The Standard Curve. set-up:

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lab 3

LAB 3

The Spectrophotometer: Measuring Concentration Using Absorbance

measuring absorbance using a spec
Measuring Absorbance using a Spec
  • set-up:
    • molar mass of KMnO4 = 158.03g
    • 0.1mg/ml KMnO4 solution = 0.63mM
determining concentration from absorbance the standard curve
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

unknown
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
beer s law
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
molar absorptivity
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

kmno4 standard curve and unknown
KMnO4 Standard Curve and Unknown

slope = 0.386 – 0.088

0.1-0.025

slope = 3.973

how d we do
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