lecture 2 spectroscopy

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SPECTROSCOPY
PHAM VAN HUNG, PhD
INTRODUCTION
• The study how the chemical compound interacts
with different wavelenghts in a given region of
electromagnetic radiation is called spectroscopy
or spectrochemical analysis.
• The collection of measurements signals
(absorbance) of the compound as a function of
electromagnetic radiation is called a spectrum.
Spectroscopy
Utilises the Absorption and Emission of electromagnetic
radiation by atoms
Absorption:
Low energy electrons absorb energy to move to higher energy level
Emission:
Excited electrons return to lower energy states
Absorption vs. Emission
Ground State
1st
2nd
3rd
Energy is absorbed as
electrons jump to
higher energy levels
Energy is emitted by
electrons returning to
lower energy levels
Excited
States

Spectroscopic Techniques
• UV-Visible Spectroscopy (UV-Vis).
• Infrared Spectroscopy (IR)
• Atomic Absorption Spectroscopy (AAS).
• Colorimetry.
UV radiation and Electronic Excitations
• The difference in energy between molecular bonding, non-
bonding and anti-bonding orbitals ranges from 125-650
kJ/mole
• This energy corresponds to electromagnetic radiation in the
ultraviolet (UV) region, 100-350 nm, and visible (VIS)
regions 350-700 nm of the spectrum
• For comparison, recall the electromagnetic spectrum:
• Using IR we observed vibrational transitions with energies
of 8-40 kJ/mol at wavelengths of 2500-15,000 nm
UVX-rays
IR
γ-rays
RadioMicrowave
Visible
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X-ray:
core electron
excitation
UV:
valance
electronic
excitation
IR:
molecular

vibrations
Radio waves:
Nuclear spin states
(in a magnetic field)
Electronic Excitation by UV/Vis Spectroscopy :
3-D structure AnaylysisX-raysX-ray Crystallography
Elemental Analysis X-raysX-Ray Spectroscopy
Structure determinationRadio wavesFT-NMR
Functional Group
Analysis/quantIR/UVRaman
Functional Group AnalysisIR/MicrowaveFT-IR
Quantitative analysis
Beer’s LawUV-vis regionAtomic Absorption
Quantitative
analysis/Beer’s LawUV-vis regionUV-vis
Spectroscopic Techniques and Common Uses
Different Spectroscopies
• UV/Vis – electronic states of valence e/d-orbital
transitions for solvated transition metals
• Fluorescence – emission of UV/vis by certain
molecules
• FT-IR – vibrational transitions of molecules
• FT-NMR – nuclear spin transitions
• X-Ray Spectroscopy – electronic transitions of
core electrons
Dispersion of Polymagnetic Light with a Prism
Polychromatic
Ray
Infrared
Red

Orange
Yellow
Green
Blue
Violet
Ultraviolet
monochromatic
Ray
SLIT
PRISM
Polychromatic Ray Monochromatic Ray
• Prism - Spray out the spectrum and choose the certain wavelength (λ) that
you want by slit.
• In UV spectroscopy, the sample is irradiated with the broad spectrum of the
UV radiation
• If a particular electronic transition matches the energy of a certain band of
UV, it will be absorbed
Electronic Excitation
The absorption of light energy by organic compounds in the
visible and ultraviolet region involves the promotion of
electrons in σ, π, and n-orbitals from the ground state to higher
energy states. This is also called energy transition. These higher
energy states are molecular orbitals called antibonding.
Energy
σ
*
π
*
n

π

σ
σ
→σ*
π→π*
n
→σ*
n →π*
Antibonding
Antibonding
N
onbonding
Bonding
Bonding
Electronic Molecular Energy Levels
Energy
σ
*
π
*
n

π

σ
σ
→σ*
π→π*
n

→σ*
n
→π
*
Antibonding
Antibonding
N
onbonding
Bonding
Bonding
• For any bond (pair of electrons) in a molecule, the molecular orbitals are a mixture of the
two contributing atomic orbitals; for every bonding orbital “created” from this mixing (s, p),
there is a corresponding anti-bonding orbital of symmetrically higher energy (s*, p*).
• The lowest energy bonding orbitals are typically the s; likewise, the corresponding anti-
bonding s* orbital is of the highest energy.
• p-orbitals are of somewhat higher energy, and their complementary anti-bonding orbital
somewhat lower in energy than s*.
• Unshared pairs lie at the energy of the original atomic orbital, most often this energy is
higher than p or s (since no bond is formed, there is no benefit in energy).
• The higher energy transitions (σ→σ*) occur a shorter wavelength and the low energy
transitions (π→π*, n →π*) occur at longer wavelength.
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Observed electronic transitions
From the molecular orbital diagram, there are several possible electronic
transitions that can occur, each of a different relative energy:
Energy
σ∗
π
σ
π∗

n
σ
σ
π
n
n
σ
∗
π
∗
π
∗
σ
∗
π
∗
alkanes
carbonyls
unsaturated cmpds.
O, N, S, halogens
carbonyls
Observed electronic transitions
- Routine organic UV spectra are typically collected from 200-700 nm
- This limits the transitions that can be observed:
σ
σ
π
n
n
σ

∗
π
∗
π
∗
σ
∗
π
∗
alkanes
carbonyls
unsaturated cmpds.
O, N, S, halogens
carbonyls
150 nm
170 nm
180 nm √ - if conjugated!
190 nm
300 nm √
UV
210 nm Double Bonds
233 nm Conjugated Diene
268 nm Conjugated Triene
315 nm Conjugated Tetraene
Observed electronic transitions
- Remember the electrons present in organic molecules are involved
in covalent bonds or lone pairs of electrons on atoms such as O or N
- A functional group capable of having characteristic electronic
transitions is called a chromophore (color loving).
- Chromophore is a functional group which absorbs a characteristic

ultraviolet or visible region.
CC
CC
CO
CO
H
σ −> σ∗ 135 nm
π −> π∗ 165 nm
n −> σ∗ 183 nm weak
π −> π∗ 150 nm
n −> σ∗ 188 nm
n −> π∗ 279 nm weak
λ
A
180 nm
279 nm
CO
Spectrum
Spectrum
Glass cell filled with
concentration of solution (C)
I
I
Light
0
Transmittance is defined as the ratio of the electromagnetic radiation’s power
exiting the sample, I, to that incident on the sample from the source, I
0
,
I

I
0
T =
An alternative method for expressing the attenuation of electromagnetic
radiation is absorbance, A, which is defined as
A = - Log T = - Log = Log
I
0
I
I
I
0
Transmittance and Absorbance
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Beer – Lambert Law
• There is a logarithmic dependence between the transmission, T, of light
through a substance and the product of the absorption coefficient
of the
substance, α, and the distance the light travels through the material, ℓ.
• The absorption coefficient
can, in turn, be written as a product of either a
molar absorptivity
(extinction coefficient) of the absorber, ε, and the molar
concentration c of absorbing species in the material.
• The molar absorptivity give, in effect, the probability that the analyte will absorb
a photon of given energy. As a result, value for
ε
depend on the wavelength of
electromagnetic radiation. Compound x has a unique e at different wavelengths.
• Unit of

ε
: L*cm
-1
*M
-1
Steps in Developing a Spectrometric Analytical Method
1. Run the sample for spectrum
2. Obtain a monochromatic
wavelength for the maximum
absorption wavelength.
3. Calculate the concentration of
your sample using Beer Lambert
Equation: A =
ε
lc
Wavelength (nm)
Absorbance
0.0
2.0
200
250 300
350
400
450
Spectrophotometer
An instrument which can measure the absorbance of a
sample at any wavelength.
Light Lens
Slit Monochromator
Sample Detector

Quantitative Analysis
Slits
Instrument to measures the intensity of fluorescent light emitted by a sample
exposed to UV light under specific conditions.
Emit fluorescent light
as energy decreases
Ground state
Sample
90
°
C
Detector
UV Light Source
Monochromator
Monochromator
Antibonding
Antibonding
Nonbonding
Bonding
Bonding
Energy
σ
π
σ
π
σ −>σ
π −>π
'
'
'

'
'
n->
n
σ
n->π
'
Electron's molecular energy levels
Fluorometer
The optics of the light source in UV-visible spectroscopy
allow either visible [approx. 400nm (blue end) to 750nm
(red end) ] or ultraviolet (below 400nm) to be directed at
the sample under analysis (common range: 200 – 800 nm).
UV/Vis Spectrophotometer
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Cuvette
UV Spectrophotometer
Quartz (crystalline silica)
Visible Spectrophotometer
Glass, Plastic
Light Sources
UV Spectrophotometer
1. Deuterium (200-400 nm)
Visible Spectrophotometer
1. Tungsten Lamp (350-2500 nm)
UV Spectrometer Application
Protein
Amino Acids (aromatic)
Pantothenic Acid
Glucose Determination

Enzyme Activity (Hexokinase)
Visible Spectrometer Application
Niacin
Pyridoxine
Vitamin B12
Metal Determination (Fe)
Fat-quality Determination (TBA)
Enzyme Activity (glucose oxidase)
Flurometric Application
Thiamin (365 nm, 435 nm)
Riboflavin
Vitamin A
Vitamin C
Standard Practice
• Prepare standards of known concentration
• Measure absorbance at λmax of solution
at different concentration
• Plot A vs. concentration
• Obtain slope
• Use slope (and intercept) to determine the
concentration of the analyte in the
unknown
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Typical Beer’s Law Plot
y = 0.02x
0
0.2
0.4
0.6
0.8

1
1.2
0.0 20.0 40.0 60.0
concentration (uM)
A
R
2
= 0.995
Characteristics of Beer’s Law Plots
• One wavelength
• Good plots have a range of absorbances from
0.010 to 1.000
• Absorbances over 1.000 are not that valid and
should be avoided
Chromophoric Structure
Group Structure nm
Carbonyl > C = O 280
Azo -N = N- 262
Nitro -N=O 270
Thioketone -C =S 330
Nitrite -NO2 230
Conjugated Diene -C=C-C=C- 233
Conjugated Triene -C=C-C=C-C=C- 268
Conjugated Tetraene -C=C-C=C-C=C-C=C- 315
Benzene 261
Practice Examples
1. Calculate the Molar Extinction Coefficient E at 351 nm for
aquocobalamin in 0.1 M phosphate buffer. pH = 7.0 from the
following data which were obtained in 1 Cm cell.
Solution

C x 10
5
M Io I
A 2.23 100 27
B 1.90 100 32
2. The molar extinction coefficient (E) of compound
riboflavin is 3 x 10
3
Liter/Cm x Mole. If the absorbance
reading (A) at 350 nm is 0.9 using a cell of 1 Cm, what is the
concentration of compound riboflavin in sample?
3. The concentration of compound Y was 2 x 10
-4
moles/liter and
the absorption of the solution at 300 nm using 1 Cm quartz cell
was 0.4. What is the molar extinction coefficient of compound
Y?
4. Calculate the molar extinction coefficient E at 351 nm for
aquocobalamin in 0.1 M phosphate buffer. pH =7.0 from the
following data which were obtained in 1 Cm cell.
Solution
C x 10
5
M I0 I
A 2.0 100 30
Spectroscopy Homework
1. A substance absorbs at 600 nm and 4000 nm. What type of energy
transition most likely accounts for each of these absorption
processes?
2. Complete the following table.

[X](M) Absorbance Transmittance(%) E(L/mole-cm) L(cm)
30 2000 1.00
0.5 2500 1.00
2.5 x 10
-3
0.2 1.00
4.0 x 10
-5
50 5000
2.0 x 10
-4
150
[X](M) = Concentration in Mole/L
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3. The molar absorptivity of a pigment (molecular weight 300)
is 30,000 at 550 nm. What is the absorptivity in L/g-cm.
4. The iron complex of o-phenanthroline (Molecular weight
236) has molar absorptivity of 10,000 at 525 nm. If the
absorbance of 0.01 is the lowest detectable signal, what
concentration in part per million can be detected in a 1-cm
cell?