spectrophotometry: an analytical tool
DESCRIPTION
Spectrophotometry: An Analytical Tool. The process of light being absorbed by a solution. concentration 2. with sample I < I o. concentration 1. blank where I o = I. light source. detector. I o. I. As concentration increases, less light is transmitted (more light absorbed). b. - PowerPoint PPT PresentationTRANSCRIPT
Spectrophotometry:
An Analytical Tool
PGCC CHM 103 Sinex/Gage
Io I
Cell withPathlength, b,
containing solution
lightsource detector
blank where Io = I
concentration 2concentration 1
b
with sample I < Io
The process of light being absorbed by a solution
As concentration increases, less light is transmitted (more light absorbed).
PGCC CHM 103 Sinex/Gage
Some terminologyI – 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
Remembering the “More Lights, Color, Absorption” lab activity, what factors affect the amount of light that is absorbed by a solution in a spectrometer?
PGCC CHM 103 Sinex/Gage
PGCC CHM 103 Sinex/Gage
Beer’s Law
A = abcwhere
a = molar absorptivity (actually the symbol ε is the correct symbol for this but a is
easier to remember)
b = pathlengthc = molar concentration
See the Beer’s Law Simulator
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))
PGCC CHM 103 Sinex/Gage
PGCC CHM 103 Sinex/Gage
Analyze at what wavelength?
Scan visible wavelengths from 400 – 650 nm (detector range) to produce an
absorption spectrum (A vs. )Crystal Violet Absorption Spectrum
0
0.2
0.4
0.6
0.8
1
1.2
1.4
200 250 300 350 400 450 500 550 600 650 700 750wavelength, nm
Abso
rban
ce
max
max - wavelength where maximum absorbance occurs
phototube detector range
PGCC CHM 103 Sinex/Gage
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.
PGCC CHM 103 Sinex/Gage
Light source
Grating
Rotating the gratingchanges the wavelength going through the sample
slits
slits
Sample
filter
Phototube
The components of a Spec-20D
occluder
When blank is the sample Io is determined
otherwise I is measured
Separates white lightinto various colors
detects light &measures intensity
- white light of constant intensity
PGCC CHM 103 Sinex/Gage
What does the absorbed light (electromagnetic radiation)
do to the molecule?
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)
700 nm 400 nm
IR UV
visibleEnergy increasing
PGCC CHM 103 Sinex/Gage
UV/visible light absorption
In organic molecules, electronic transitions to higher energy molecular orbitals – double bonds: *
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)4
2+ – strong absorption
Valence electrons
PGCC CHM 103 Sinex/Gage
Uses of visible spectrophotometry
Analysis of unknowns using Beer’s Law calibration curve
Absorbance vs. time graphs for kineticsSingle-point calibration for an equilibrium
constant determinationSpectrophotometric 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!)
Standard Curves
PGCC CHM 103 Sinex/Gage
Concentration (mol/L or M)0.01 0.02 0.05 0.06 0.070.03 0.04
Absorbance
regression equation
If you know the absorbance of an unknown youcan determine the concentration.
PGCC CHM 103 Sinex/Gage
Kinetics of Crystal Violet Reaction
CV+ + OH- CV-OHpurple 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
PGCC CHM 103 Sinex/Gage
ab
sorb
an
ce
time
Since the absorbance is related to concentration, rate or A/time is the slope of a regression line.
CV+ + OH- CV-OHpurple colorless colorless
Short run times to get initial rates.
STELLA model
This is tracking reaction progress over time.
PGCC CHM 103 Sinex/Gage
Single-point calibration• Standard with measured absorbance
Astd = abcstd
• Unknown with measured absorbanceAunk = abcunk
Ratio the two equationsAunk/ Astd = abcunk /abcstd
Aunk/ Astd = cunk /cstd
• Solve for cunk
PGCC CHM 103 Sinex/Gage
Equilibrium Constant Determination
Fe+3 + 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
PGCC CHM 103 Sinex/Gage
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
Calibration Curve
0
0.2
0.4
0.6
0.8
1
0 0.2 0.4 0.6 0.8 1concentration
Abso
rban
ce
linear
curved
Linear (linear)
Non-linear
PGCC CHM 103 Sinex/Gage
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?
PGCC CHM 103 Sinex/Gage
abso
rban
ce
Volume of titrant (mL KMnO4)
5H2O2 + 2MnO4- + 6H+ 5O2 (g) + 2Mn+2 + 8H2O
purple
Equivalence point
MnO4- reacting,
color disappears xs MnO4-
accumulates
Notice you do not need to have adata point at the equivalence point. Equivalence point located by extrapolation of the two lines.