CHM 103
Sinex
The process of light being absorbed by a solution
concentration 2
concentration 1
blank where Io = I
Spectrophotometry:
light
source
An Analytical Tool
detector
Io
I
b
PGCC CHM 103 Sinex
Cell with
Pathlength, b,
containing solution
Some terminology
Beer’s Law
ØI – intensity where Io is initial intensity
A = abc
ØT – transmission or %T = 100 x T
(absorption: Abs = 1 – T or %Abs = 100 - %T)
T = I/ Io
with sample
I < Io
As concentration
increased, less
light was
transmitted (more
light absorbed).
where a – molar absorptivity, b – pathlength,
and c – molar concentration
See the Beer’s Law Simulator
ØA – absorbance
A = - log T = -log I/ Io
PGCC CHM 103 Sinex
PGCC CHM 103 Sinex
Analyze at what wavelength?
Scan visible wavelengths from 400 – 650 nm
(detector range) to produce an absorption
spectrum (A vs. λ)
Crystal Violet Absorption Spectrum
1.4
Absorbance
1.2
1
0.8
0.6
λmax
0.4
0.2
0
200
250
300
350
400
450
500
wavelength, nm
550
600
650
phototube detector range
700
750
λmax - wavelength where maximum absorbance occurs
PGCC CHM 103 Sinex
The BLANK
üThe blank contains all substances
expect the analyte.
üIs used to set the absorbance to zero:
Ablank = 0
üThis removes any absorption of light
due to these substances and the cell.
üAll measured absorbance is due to
analyte.
PGCC CHM 103 Sinex
1
CHM 103
Sinex
What does the absorbed light
(electromagnetic radiation)
do to the molecule?
The components of a Spec-20D
Light source - white light of constant intensity
IR
slits
filter
Separates white light
into various colors
slits
Sample
When blank is the sample
Io is determined
PGCC CHM 103 otherwise
Sinex
I is measured
Rotating the grating
changes the wavelength
going through the sample
Ø low energy UV and visible – promotes electrons
to higher energy orbitals
(absorption of visible light leads to a colored solution)
Ø IR – causes molecules to vibrate (more later)
PGCC CHM 103 Sinex
Uses of visible
spectrophotometry
UV/visible light absorption
Valence electrons
Ø In organic molecules, electronic transitions to
higher energy molecular orbitals – double
bonds: π à π*
Ø In transition metals, hydrated ions as Cu++
have splitting of d orbital energies and
electronic transitions – weak absorption
Ø In complexed transition metals, charge
transfer of electrons from metal to ligand as
Cu(NH3)4++ – strong absorption
PGCC CHM 103 Sinex
ü Analysis of unknowns using Beer’s Law
calibration curve
(Been there, done that!)
ü Absorbance vs. time graphs for kinetics
ü Single-point calibration for an equilibrium
constant determination
ü 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
PGCC CHM 103 Sinex
CV+
+
OHà CV-OH
purple
colorless
colorless
Kinetics of Crystal Violet Reaction
This is tracking reaction progress over time.
colorless
colorless
Follow concentration of crystal violet over
time as it reacts by measuring its absorbance.
Since the absorbance is
related to concentration,
rate or ∆A/∆time is the
slope of a regression line.
absorbance
CV+ + OH- à CV-OH
purple
UV
400 nm
Ø high energy UV – ionizes electrons
occluder
Grating
Phototube
detects light &
measures intensity
visible
Energy increasing
700 nm
How will absorbance change with time?
For a absorbance vs. time plot, how will you
determine the rate of the reaction?
PGCC CHM 103 Sinex
Chime structures
Short run times to get initial rates.
time
PGCC CHM 103 Sinex
STELLA model
2
CHM 103
Sinex
Single-point calibration
Equilibrium Constant Determination
Fe+3
• Standard with measured absorbance
Astd = abcstd
• Unknown with measured absorbance
Aunk = abcunk
colorless
Fe(SCN)++ + SCN- = Fe(SCN)2+
We start with a high concentration of Fe+3
and lower its value by dilution.
PGCC CHM 103 Sinex
When calibration curves go bad!
• The linear Beer’s Law relationship starts to
show curvature at high concentrations
Absorbance
Calibration Curve
0.8
Non-linear
linear
curved
0.4
0.2
Linear (linear)
0
0
0.2
0.4
0.6
concentration
0.8
1
• Single-point calibration assumes a linear
calibration curve
PGCC CHM 103 Sinex
absorbance
5H2O 2
orange
Using the reactants, shift reaction based
on Le Chatelier’s principle.
PGCC CHM 103 Sinex
1
colorless
K = (Fe(SCN)++)/(Fe+3 )(SCN-)
Ratio the two equations
Aunk/ Astd = abcunk /abcstd
Aunk/ Astd = cunk /cstd
• Solve for cunk
0.6
+ SCN- = Fe(SCN)++
Interactive Excel spreadsheet
Spectrophotometric titration
• Let’s consider the analysis of hydrogen
peroxide with potassium permanganate
in an acidic solution.
• The potassium permanganate or MnO 4is the only colored substance in the
reaction. (It can serve as its own
indicator.)
• How would the absorbance change as
titrant was added?
PGCC CHM 103 Sinex
+ 2MnO 4- + 6H+
purple
à 5O 2 (g) +
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
MnO 4- reacting,
color disappears
PGCC CHM 103 Sinex
xs MnO 4accumulates
Volume of titrant (mL KMnO 4 )
3