1. Spectrophotometry: An Analytical Tool
CHM 103
Sinex
Spectrophotometry: An Analytical Tool

2. Io I The process of light being absorbed by a solution concentration 2
with sample
I < Io
concentration 1
blank where Io = I
light
source
detector Io I
As concentration increases, less light is transmitted (more light absorbed). b
Cell with
Pathlength, b,
containing solution
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3. Some terminology
I – intensity where Io is initial intensity of light entering a solution and I is the intensity of light exiting a solution
T – transmission (no units, ratio)
T = I/ Io %T = 100 x T
(absorption: Abs = 1 – T or
%Abs = %T)
A – absorbance (no units)
A = - log T = -log I/ Io
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4. Remembering the “More Lights, Color, Absorption” lab activity, what factors affect the amount of light that is absorbed by a solution in a spectrometer?
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5. A = abc Beer’s Law a = molar absorptivity b = pathlength
where
a = molar absorptivity
(actually the symbol ε is the correct symbol for this but a is easier to remember)
b = pathlength
c = molar concentration
See the Beer’s Law Simulator
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6. Molar absorptivity
Depends on the electronic structure of the substance being analyzed (analyte)
Varies with the wavelength of light because a compound absorbs different amounts at different wavelengths
Units = L mol-1 cm-1
(a = A/bc = 1/(mol/L x cm))
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7. Analyze at what wavelength?
Scan visible wavelengths from 400 – 650 nm (detector range) to produce an absorption spectrum (A vs. l)
lmax
phototube detector range
lmax - wavelength where maximum absorbance occurs
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8. The BLANK The blank contains all substances except the analyte.
Is used to set the absorbance to zero:
Ablank = 0
This removes any absorption of light due to these substances and the cell.
All measured absorbance is due to analyte.
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9. The components of a Spec-20D
Light source
- white light of constant intensity
slits
filter
occluder
Grating
slits
Separates white light
into various colors
Phototube
detects light &
measures intensity
Rotating the grating
changes the wavelength going through the sample
Sample
When blank is the sample
Io is determined
otherwise I is measured
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10. What does the absorbed light (electromagnetic radiation) do to the molecule?IR UV
700 nm
visible
400 nm
Energy increasing
high energy UV – ionizes electrons
low energy UV and visible – promotes electrons to higher energy orbitals
(absorption of visible light leads to a colored solution)
IR – causes molecules to vibrate (more later)
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11. UV/visible light absorption
Valence electrons
In organic molecules, electronic transitions to higher energy molecular orbitals – double bonds: p  p*
In transition metals, hydrated ions such as Cu2+ have splitting of d orbital energies and electronic transitions – weak absorption
In complexed transition metals, charge transfer of electrons from metal to ligand as Cu(NH3)42+ – strong absorption
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12. Uses of visible spectrophotometry
Analysis of unknowns using Beer’s Law calibration curve
Absorbance vs. time graphs for kinetics
Single-point calibration for an equilibrium constant determination
Spectrophotometric titrations – a way to follow a reaction if at least one substance is colored – sudden or sharp change in absorbance at equivalence point, a piece-wise function
(Been there, done that!)
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13. Standard Curves Concentration (mol/L or M) 0.01 0.02 0.05 0.06 0.07
0.03
0.04
Absorbance
regression equation
If you know the absorbance of an unknown you
can determine the concentration.
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14. purple colorless colorless
Kinetics of Crystal Violet Reaction
CV OH-  CV-OH
purple colorless colorless
Follow concentration of crystal violet over time as it reacts by measuring its absorbance.
How will absorbance change with time?
For a absorbance vs. time plot, how will you determine the rate of the reaction?
Chime structures
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15. purple colorless colorless
CV OH  CV-OH
purple colorless colorless
This is tracking reaction progress over time.
Since the absorbance is
related to concentration,
rate or DA/Dtime is the
slope of a regression line.
absorbance
Short run times to get initial rates.
time
STELLA model
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16. Single-point calibration
Standard with measured absorbance
Astd = abcstd
Unknown with measured absorbance
Aunk = abcunk
Ratio the two equations
Aunk/ Astd = abcunk /abcstd
Aunk/ Astd = cunk /cstd
Solve for cunk
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17. Equilibrium Constant Determination
Fe SCN- = Fe(SCN)++
colorless colorless orange
K = (Fe(SCN)++)/(Fe+3)(SCN-)
Using the reactants, shift reaction based on Le Chatelier’s principle.
Fe(SCN)++ + SCN- = Fe(SCN)2+
We start with a high concentration of Fe+3 and lower its value by dilution.
Interactive Excel spreadsheet
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18. When calibration curves go bad!
The linear Beer’s Law relationship starts to show curvature at high concentrations
Single-point calibration assumes a linear calibration curve
Non-linear
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19. Spectrophotometric titration
Let’s consider the analysis of hydrogen peroxide with potassium permanganate in an acidic solution.
The potassium permanganate or MnO4- is the only colored substance in the reaction. (It can serve as its own indicator.)
How would the absorbance change as titrant was added?
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20. 5H2O2 + 2MnO4- + 6H+  5O2 (g) + 2Mn+2 + 8H2O purple
Notice you do not need to have a
data point at the equivalence point.
Equivalence point located by extrapolation of the two lines.
absorbance
Equivalence point
MnO4- reacting,
color disappears
xs MnO4-
accumulates
Volume of titrant (mL KMnO4)
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