CHM 103 Sinex

The process of light being absorbed by a solution

concentration 2 with sample
concentration 1
I < Io
blank where Io = I

Spectrophotometry:
light
An Analytical Tool source detector

Io I
As concentration
b increased, less
light was
Cell with
transmitted (more
Pathlength, b,
light absorbed).
PGCC CHM 103 Sinex containing solution

Some terminology Beer’s Law

ØI – intensity where Io is initial intensity
A = abc
ØT – transmission or %T = 100 x T where a – molar absorptivity, b – pathlength,
(absorption: Abs = 1 – T or %Abs = 100 - %T) and c – molar concentration
T = I/ Io
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? The BLANK

Scan visible wavelengths from 400 – 650 nm
(detector range) to produce an absorption üThe blank contains all substances
spectrum (A vs. λ)
expect the analyte.
Crystal Violet Absorption Spectrum
1.4

1.2
üIs used to set the absorbance to zero:
1
Ablank = 0
Absorbance

0.8

0.6 üThis removes any absorption of light

0.4
λmax due to these substances and the cell.
0.2

0
200 250 300 350 400 450 500 550 600 650 700 750 üAll measured absorbance is due to
wavelength, nm
phototube detector range analyte.
λmax - wavelength where maximum absorbance occurs
PGCC CHM 103 Sinex PGCC CHM 103 Sinex

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CHM 103 Sinex

What does the absorbed light

The components of a Spec-20D
(electromagnetic radiation)
Light source - white light of constant intensity do to the molecule?
IR UV
slits 700 nm visible 400 nm
Energy increasing

filter occluder Ø high energy UV – ionizes electrons

Grating Ø low energy UV and visible – promotes electrons
Phototube
slits Separates white light to higher energy orbitals
into various colors
detects light & (absorption of visible light leads to a colored solution)
measures intensity Rotating the grating
Sample
changes the wavelength
When blank is the sample going through the sample Ø IR – causes molecules to vibrate (more later)
Io is determined
PGCC CHM 103 otherwise
Sinex I is measured PGCC CHM 103 Sinex

Uses of visible
UV/visible light absorption
spectrophotometry
Valence electrons
Ø In organic molecules, electronic transitions to ü Analysis of unknowns using Beer’s Law
higher energy molecular orbitals – double calibration curve (Been there, done that!)

bonds: π à π* ü Absorbance vs. time graphs for kinetics

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 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+ + OH- à CV-OH

Kinetics of Crystal Violet Reaction purple colorless colorless
This is tracking reaction progress over time.
CV+ + OH- à CV-OH
purple colorless colorless
Since the absorbance is
absorbance

related to concentration,
Follow concentration of crystal violet over rate or ∆A/∆time is the
time as it reacts by measuring its absorbance. slope of a regression line.

How will absorbance change with time?

For a absorbance vs. time plot, how will you

Short run times to get initial rates.
determine the rate of the reaction?

time
PGCC CHM 103 Sinex Chime structures PGCC CHM 103 Sinex STELLA model

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CHM 103 Sinex

Single-point calibration Equilibrium Constant Determination

• Standard with measured absorbance Fe+3 + SCN- = Fe(SCN)++
colorless colorless orange
Astd = abcstd
• Unknown with measured absorbance K = (Fe(SCN)++)/(Fe+3 )(SCN-)
Aunk = abcunk
Using the reactants, shift reaction based
Ratio the two equations on Le Chatelier’s principle.

Aunk/ Astd = abcunk /abcstd Fe(SCN)++ + SCN- = Fe(SCN)2+

Aunk/ Astd = cunk /cstd We start with a high concentration of Fe+3
• Solve for cunk and lower its value by dilution.
PGCC CHM 103 Sinex PGCC CHM 103 Sinex Interactive Excel spreadsheet

When calibration curves go bad! Spectrophotometric titration

• The linear Beer’s Law relationship starts to • Let’s consider the analysis of hydrogen
show curvature at high concentrations peroxide with potassium permanganate
1 Calibration Curve in an acidic solution.
0.8
Non-linear
Absorbance

0.6
linear
• The potassium permanganate or MnO 4-
is the only colored substance in the
0.4 curved
0.2 Linear (linear)
0
0 0.2 0.4 0.6 0.8 1
reaction. (It can serve as its own
concentration
indicator.)
• Single-point calibration assumes a linear • How would the absorbance change as
calibration curve titrant was added?
PGCC CHM 103 Sinex PGCC CHM 103 Sinex

5H2O 2 + 2MnO 4- + 6H+ à 5O 2 (g) + 2Mn+2 + 8H2O

purple

Notice you do not need to have a

data point at the equivalence point.
Equivalence point located by
absorbance

extrapolation of the two lines.

Equivalence point

MnO 4- reacting,
color disappears xs MnO 4-
accumulates

Volume of titrant (mL KMnO 4 )

PGCC CHM 103 Sinex