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:
–
–
–
–
–
–
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
64-fold
dilution
0.1mg/ml
2.5 ml water for dilution
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
KMnO4 Standard Curve
Column1
Column2
Column3
0.1mg/ml KMnO4
Sample # [KMnO4] mg/ml Abs545nm
1
0.1
2
0.05
3
0.025
4
0.0125
5
0.00625
6
0.003125
7
0.0015625
Column4
Column5
0.386
0.213
0.088
0.049 Unknown #1
0.012 Unknown #2
-0.015 Unknown #3
0.017
0.201
0.943
0.6
KMnO4 Absorbance vs. Concentration
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
-0.05
0.00156
0.00312
0.00625
0.0125
0.025
0.05
0.1
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 (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
c
unknown
=A
/eb
unknown
-b is 1cm
-units are whatever the
units are for the standards
KMnO4 Standard Curve and Unknown
Column1
Column2
Column3
0.1mg/ml KMnO4
Sample # [KMnO4] mg/ml Abs545nm
1
0.1
2
0.05
3
0.025
4
0.0125
5
0.00625
6
0.003125
7
0.0015625
slope
Column4
Column5
0.386
0.213
0.088
0.049 Unknown #1
0.012 Unknown #2
-0.015 Unknown #3
0.017
0.201
0.943
0.6
slope = 0.386 – 0.088
0.1-0.025
slope = 3.973
KMnO4 Absorbance vs. Concentration
3.973333333
0.45
unknown #1
concentration
0.201
0.0505 mg/ml
unknown #2
concentration
0.943
0.2373 mg/ml
unknown #3
concentration
0.6
0.1510 mg/ml
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
-0.05
0.00156
0.00312
0.00625
0.0125
0.025
0.05
0.1
How’d we do?
• 0.1 mg/ml KMnO4 is 0.63 mM
• 158.03 mg in 1 ml of water is 1M KMnO4
• 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