Spectrophotometry:
An Analytical Tool
The process of light being absorbed by a solution
concentration 2
concentration 1
blank where Io = I
light
source
with sample
I < Io
detector
Io
I
b
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Cell with
Pathlength, b,
containing solution
As concentration
increases, less
light is
transmitted (more
light absorbed).
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 = 100 - %T)
A – absorbance (no units)
A = - log T = -log I/ Io
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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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Beer’s Law
A = abc
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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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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Analyze at what wavelength?
Scan visible wavelengths from 400 – 650 nm
(detector range) to produce an absorption
spectrum (A vs. l)
Crystal Violet Absorption Spectrum
1.4
Absorbance
1.2
1
0.8
0.6
lmax
0.4
0.2
0
200
250
300
350
400
450
500
wavelength, nm
550
600
650
phototube detector range
700
750
lmax - wavelength where maximum absorbance occurs
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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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The components of a Spec-20D
Light source - white light of constant intensity
slits
filter
occluder
Grating
Phototube
detects light &
measures intensity
slits
Sample
When blank is the sample
Io is determined
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Separates white light
into various colors
Rotating the grating
changes the wavelength
going through the sample
IR
What does the absorbed light
(electromagnetic radiation)
do to the molecule?
700 nm
visible
Energy increasing
UV
400 nm
 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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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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Uses of visible
spectrophotometry
 Analysis of unknowns using Beer’s Law
calibration curve (Been there, done that!)
 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
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Standard Curves
A
b
s
o
r
b
a
n
c
e
regression equation
0.01
0.02
0.03
0.04
0.05
Concentration (mol/L or M)
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0.06
0.07
If you know the absorbance of an unknown you
can determine the concentration.
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?
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Chime structures
CV+ + OH CV-OH
purple colorless
colorless
This is tracking reaction progress over time.
absorbance
Since the absorbance is
related to concentration,
rate or DA/Dtime is the
slope of a regression line.
Short run times to get initial rates.
time
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STELLA model
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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Equilibrium Constant Determination
Fe+3
colorless
+ SCN- = Fe(SCN)++
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.
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Interactive Excel spreadsheet
When calibration curves go bad!
• The linear Beer’s Law relationship starts to
show curvature at high concentrations
Absorbance
1
Calibration Curve
0.8
Non-linear
0.6
linear
curved
Linear (linear)
0.4
0.2
0
0
0.2
0.4
0.6
concentration
0.8
1
• Single-point calibration assumes a linear
calibration curve
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Spectrophotometric titration
• Let’s consider the analysis of hydrogen
peroxide with potassium permanganate
in an acidic solution.
• The potassium permanganate or MnO4is 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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absorbance
5H2O2 + 2MnO4- + 6H+  5O2 (g) +
purple
2Mn+2 + 8H2O
Notice you do not need to have a
data point at the equivalence point.
Equivalence point located by
extrapolation of the two lines.
Equivalence point
MnO4- reacting,
color disappears
xs MnO4accumulates
Volume of titrant (mL KMnO4)
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