Molecular Spectroscopy

Visible and Ultraviolet Spectroscopy

－ UV/VIS Spectroscopy
－ UV/VIS Spectrometer
－ Application for Quantitative
Analysis
• Ultraviolet: 190~400nm
• Violet:   400 - 420 nm
• Indigo:   420 - 440 nm
• Blue:   440 - 490 nm
• Green:   490 - 570 nm
• Yellow:   570 - 585 nm
• Orange:   585 - 620 nm
• Red:   620 - 780 nm
 The theory
• Visible light lies in the wavelength range 4.0 –
400–700 nm.
• When light is absorbed by a material, valence
(outer) electrons are promoted from their
normal (ground) states to higher energy
(excited) states.

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 Electronic Spectroscopy
• Ultraviolet (UV) and visible (VIS) spectroscopy
• This is the earliest method of molecular
spectroscopy.
• A phenomenon of interaction of molecules with
ultraviolet and visible lights.
• Absorption of photon results in electronic
transition of a molecule, and electrons are
promoted from ground state to higher
electronic states.
 UV and Visible Spectroscopy

• In structure determination : UV-VIS

spectroscopy is used to detect the presence of
chromophores like dienes, aromatics,
polyenes, and conjugated ketones, etc.
 Terms describing UV absorptions

1.  Chromophores: functional groups that give

electronic transitions.
2.  Auxochromes: substituents with unshared pair e's like
OH, NH, SH ..., when attached to π chromophore they
generally move the absorption max. to longer λ.
3. Bathochromic shift: shift to longer λ, also called red
shift.
4. Hysochromic shift: shift to shorter λ, also called blue
shift.
5. Hyperchromism: increase in ε of a band.
6. Hypochromism: decrease in ε of a band.
 The origin of the absorptions
• Valence electrons can generally be found in one of three types of electron
orbital:
1 ) single, or σ, bonding orbitals;
2 ) double or triple bonds (π bonding orbitals); and
3 ) non-bonding orbitals (lone pair electrons).
• Sigma bonding orbitals tend to be lower in energy than π bonding orbitals,
which in turn are lower in energy than non-bonding orbitals.
• When electromagnetic radiation of the correct frequency is absorbed, a
transition occurs from one of these orbitals to an empty orbital, usually an
antibonding orbital, σ* or π*

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 Electronic transitions

There are three types of electronic transition

which can be considered;
• Transitions involving p, s, and n electrons
• Transitions involving charge-transfer
electrons
• Transitions involving d and f electrons
 Absorbing species containing p, s,
and n electrons
• Absorption of ultraviolet and visible
radiation in organic molecules is restricted
to certain functional groups
(chromophores) that contain valence
electrons of low excitation energy.
 Vacuum UV or Far UV
(λ<190 nm )

UV/VIS
• The exact energy differences between the orbitals depends on the atoms present
and the nature of the bonding system.
• Most of the transitions from bonding orbitals are of too high a frequency (too
short a wavelength) to measure easily, so most of the absorptions observed
involve only π→π* , n→σ* and n→π* transitions.
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  Transitions

• An electron in a bonding s orbital is excited to

the corresponding antibonding orbital. The
energy required is large. For example,
methane (which has only C-H bonds, and can
only undergo  transitions) shows an
absorbance maximum at 125 nm. Absorption
maxima due to  transitions are not seen
in typical UV-VIS spectra (200 - 700 nm)
 n Transitions
• Saturated compounds containing atoms with
lone pairs (non-bonding electrons) are capable
of n  transitions. These transitions
usually need less energy than 
transitions. They can be initiated by light
whose wavelength is in the range 150 - 250
nm. The number of organic functional groups
with n  peaks in the UV region is small.
 n  and  Transitions
• Most absorption spectroscopy of organic
compounds is based on transitions of n or 
electrons to the  excited state.
• These transitions fall in an experimentally
convenient region of the spectrum (200 - 700
nm). These transitions need an unsaturated
group in the molecule to provide the 
electrons.
Chromophore Excitation max, nm Solvent

C=C →* 171 hexane

n→* 290 hexane

C=O
→* 180 hexane

N=O
n→* 275 ethanol
→* 200 ethanol

C-X   n→* 205 hexane

X=Br, I n→* 255 hexane
UV Spectrum of Isoprene

=>
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  
C C  
hv
= hv
=hc/
 
 
 
Example: ethylene absorbs at max = 165 nm = 10,000

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C O
 
 
n hv
n

 
 
n 
The n to * transition is at even longer wavelengths but is not as strong as  to *
transitions. It is said to be “ forbidden.”
Example:
Acetone: nmax = 188 nm ; = 1860
nmax = 279 nm ; = 15

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 Conjugated systems:

C C

LUMO

HOMO

• Preferred transition is between Highest Occupied Molecular Orbital

(HOMO) and Lowest Unoccupied Molecular Orbital (LUMO).
• Additional conjugation (double bonds) lowers the
HOMO-LUMO energy gap. 22
 Similar structures have similar UV spectra:

O
O

max = 238, 305 nm max = 240, 311 nm max = 173, 192 nm

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The valence electrons are the only ones whose energies permit them to be
excited by near UV/visible radiation.
(anti-bonding)

(anti-bonding)
Four types of transitions
*
n (non-bonding) *

n*
(bonding)
n*

(bonding)
  * transition in vacuum UV
n  * saturated compounds with non-bonding electrons
~ 150-250 nm
 ~ 100-3000 ( not strong)
n  *,   * requires unsaturated functional groups (eq. double bonds)
most commonly used, energy good range for UV/Vis
 ~ 200 - 700 nm
n  * :  ~ 10-100
  *:  ~ 1000 – 10,000
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 Absorption Characteristics of Some Common Chromophores
Chromophore Example Solvent max (nm) max Type of
transition
Alkene C6H13HC CH2 n-Heptane 177 13,000 *
Alkyne n-Heptane 178 10,000 *
C5H11C C CH3 196 2,000 _
225 160
_
Carbonyl O n-Hexane 186 1,000 n*
280 16 n*
CH3CCH3
O n-Hexane 180 Large
293 12
n*
CH3CH n*
Carboxyl O Ethanol 204 41 n*
CH3COH
Amido O Water 214 60 n*
CH3CNH2
Azo H3CN NCH3 Ethanol 339 5 n*

Nitro CH3NO2 Isooctane 280 22 n*

Nitroso C4H9NO Ethyl ether 300 100 _
665 20 n*
Nitrate C2H5ONO2 Dioxane 270 12 n*
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Factors affecting absorption
• The solvent

• Degree of conjugation

• Acid-base indicators

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 UV Spectral Nomenclature

Blue Shift (Hypsochromic) – Red Shift (Bathochromic) –

Peaks shift to shorter wavelength. Peaks shift to longer wavelength.

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 - For Compounds with Multiple Chromophores:

If isolated (more than one single bond apart)

-  are additive
-  constant

CH3CH2CH2CH=CH2 max= 184 max = ~10,000

CH2=CHCH2CH2CH=CH2 max=185 max = ~20,000

If conjugated - shifts to higher  (red shift)

1,3 butadiene: max= 217 nm ; max= 21,000

1,3,5-hexatriene max= 258 nm ; max= 35,000

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- For Compounds with Multiple Chromophores:

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 Solvent Effects - Intensity
Solvents can induce significant changes in the intensity of peaks.

Hyperchromic – Increase in absorption intensity.

Hypochromic – Decrease in absorption intensity.

Absorption characteristics of 2-methylpyridine

Solvent max max

Hexane 260 2000
Chloroform 263 4500
Ethanol 260 4000
Water 260 4000
Ethanol - HCl (1:1) 262 5200 30
 Solvent effects
• The excited states of most π→π* transitions are
more polar than their ground states because a
greater charge separation is observed in the
excited state.

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• If a polar solvent is used the dipole–dipole interaction reduces the energy of
the excited state more than the ground state, hence the absorption in a
polar solvent such as ethanol will be at a longer wavelength (lower energy,
hence lower frequency) than in a non-polar solvent such as hexane.

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 Solvent effects
• For n -> π* transition, the n state is much more easily
stabilized by polar solvent effects (H-bonds and
association), so in going from nonpolar to polar solvent
there is a blue shift or hypsochromic shift (decrease in
λmax, increase in ΔE).

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 O

n* Hypsochromic shift

methanol heptane

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Solvent Effects

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 Absorption laws
• The wavelength and amount of light that a compound absorbs depends on
its molecular structure and the concentration of the compound used.

• The concentration dependence follows Beer’s Law.

• A=bc = -log I/I0

• Where A is absorbance
•  is the molar absorbtivity with units of L mol-1 cm-1
• b is the path length of the sample (typically in cm).
• c is the concentration of the compound in solution, expressed in mol L-1

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 Quantitative Analysis
Beer’s Law
A=bc
: the molar absorptivity (L mol-1 cm-1)
b: the path length of the sample
c :the concentration of the compound in
solution, expressed in mol L-1
 Transmittance
I dI
T   kcdb
I0 I0
I dI b
I0 I 0  kc 0 db
I0 I
I I
 ln( )   kbc  2.303 log( )
I0 I0
I
  log( )   log T  A  bc
b I0
k

2.303
39
Instrumentation
 Components of a Spectrophotometer
Light Source

• Deuterium Lamps － a truly continuous

spectrum in the ultraviolet region is
produced by electrical excitation of
deuterium at low pressure.
(160nm~375nm)
• Tungsten Filament Lamps － the most
common source of visible and near
infrared radiation.
 Components of a Spectrophotometer
Monochromator
• Used as a filter: the monochromator will
select a narrow portion of the spectrum
(the bandpass) of a given source

• Used in analysis: the monochromator will

sequentially select for the detector to
record the different components
(spectrum) of any source or sample
emitting light.
 Monochromator
Czerny-Turner design
Grating
 Detector
Barrier Layer/Photovoltaic
 Principle of Barrier
Layer/Photovoltaic Detector
• This device measures the intensity of photons
by means of the voltage developed across the
semiconductor layer.
• Electrons, ejected by photons from the
semiconductor, are collected by the silver
layer.
• The potential depends on the number of
photons hitting the detector.
 Detector
Phototube
 Detector
Photomultiplier
 Principle of Photomultiplier
Detector
• The type is commonly used.
• The detector consists of a photoemissive
cathode coupled with a series of electron-
multiplying dynode stages, and usually called
a photomultiplier.
• The primary electrons ejected from the photo-
cathode are accelerated by an electric field so
as to strike a small area on the first dynode.
 Principle of Photomultiplier
Detector
• The impinging electrons strike with enough
energy to eject two to five secondary
electrons, which are accelerated to the second
dynode to eject still more electrons.
• A photomultiplier may have 9 to 16 stages,
and overall gain of 106~109 electrons per
incident photon.
 Single and Double Beam
Spectrometer
• Single-Beam: There is only one light beam or
optical path from the source through to the
detector.
• Double-Beam: The light from the source, after
passing through the monochromator, is split
into two separate beams-one for the sample
and the other for the reference.
 Path
length / cm 0 0.2 0.4 0.6 0.8 1.0

%T 100 50 25 12.5 6.25 3.125

Absorbance 0 0.3 0.6 0.9 1.2 1.5
External Standard and the
Calibration Curve
 Standard Addition Method
• Standard addition must be used whenever
the matrix of a sample changes the
analytical sensitivity of the method. In
other words, the slope of the working
curve for standards made with distilled
water is different from the same working
curve.
 Prepare the Standards

The concentration and volume of the stock

solution
added should be chosen to increase the
concentration of the unknown by about 30% in
each succeeding flask.
A  bC
bVx C x bVs Cs
A   kVx C x  kVs C s
Vt Vt
y  b  ax (a  kVs , x  Cs , b  kVx C x )
Vs C s
A  0  kVx C x   kVs C s  C x  
Vx

Cx: unknown concentration

 Limits to Beer’s Law
• Chemical Deviations
－ absorbing undergo association,
dissociation or reaction with the solvent
• Instrumental Deviations
－ non-monochromatic radiation
－ stray light
 Limits to Beer’s Law
Chemical Deviations
• high concentration － particles too close
• Average distance between ions and molecules
are diminished to the point.
• Affect the charge distribution and extent of
absorption.
• Cause deviations from linear relationship.
 Limits to Beer’s Law
Chemical Deviations
• chemical interactions － monomer-dimer
equilibria, metal complexation equilibria,
acid/base equilibria and solvent-analyte
association equilibria
The extent of such departure can be predicted
from molar absorptivities and equilibrium
constant. (see p561 ex 21-3)
 Limits to Beer’s Law
Instrumental Deviations
• non-monochromatic radiation
 Limits to Beer’s Law
Instrumental Deviations
• Stray light
(Po' + Po")
Am = log --------------
(P' + P")
 Sun Protection Factor is defined as the ratio of delayed sunburn on protected skin to
unprotected skin, where the protected skin is covered by 2mg/cm 2 of sunscreen.

MED (minimal erythema dose) = smallest dose (J/m2) of UVB that

produces a delayed sunburn

SPF 34 should protect you from burning for thirty-four times the
time of unprotected skin.

SPF is based on the physiological response in the wearer and

not based on a direct comparison of the chemical properties or
dosages of the compounds being used

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 Active Ingredients

Aminobenzoic acid 15%   Octyl salicylate 5%

Avobenzone 3%   Oxybenzone 6%
Cinoxate 3%   Padimate O 8%
Dioxybenzone 3%   Phenylbenzimidazole sulfonic acid 4%
Homosalate 15%   Sulisobenzone 10%
Menthyl anthranilate 5%   Titanium dioxide 25%
Octocrylene 10%   Trolamine salicylate 12%
Octyl methoxycinnamate 5%   Zinc oxide 25%

(FDA, 1999, p27687)

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