Molecular Spectroscopy

A Review

Goals
Review and understand what makes
a molecule absorb UV-Vis radiation
Review Beers Law and its
limitations
Review and understand Fluorescence
Review Instrument design.

Molecular Spectroscopy (Review)

Spectroscopy of the electrons surrounding an atom or a molecule: electron

energy-level transitions

Atoms: electrons are in

hydrogen-like orbitals
(s, p, d, f)

Molecules: electrons are in

molecular orbitals (HOMO,
LUMO, )

From
http://education.jlab.org

(The Bohr model for nitrogen)

(The LUMO of benzene)3

2-Particle property (light as energy)

Light consist of energy packets, known as photons.
The energy (E) of photons is proportional to the frequency i.e
related to c and . It can be expressed by max plank relation:
E=h
where h = max plank constant = 3.63 x 10-27 erg., sec.)

i.e

or

( = C / )
1/

Therefore energy of a beam of EMR increases as wave length

decreases.
4

 The shorter the wave length, the greater the energy of

the photons and the more powerful the radiation.
U.V range (200nm-400nm) which contain shorter ,
carrying more energy photon than a beam of visible
range .
Visible range (400nm-800nm) having high energy more
than I.R range (>800nm).

Molecular UV-Vis Spectroscopy:

Transitions
Major classes of electron transitions
HOMO: highest occupied molecular orbital
LUMO: lowest unoccupied molecular orbital
Types of electron transitions:
(1) , and n electrons (mostly organics)
(2) d and f electrons (inorganics/organometallics)
(3) charge-transfer (CT) electrons

For analytical purposes we use the region of

.R, visible and U.V radiations .
-UV radiation region is classified into :
far UV from (10nm-200nm) and near UV from ( 200nm-380nm-

-Visible radiation region (380-780nm) consist of colored radiations,

which are, violet, indigo, blue, green, yellow, orange and red .
-IR radiation region ( 0.75um-1000um ) is classified into :
near IR , mid IR and far IR .

Molecular UV-Vis Spectroscopy: Theory

Molecular energy levels and absorbance wavelength:

* and * transitions: high-energy, accessible in vacuum

UV (max <150 nm). Not usually observed in molecular UV-Vis.
n * and * transitions: non-bonding electrons (lone pairs),
wavelength (max) in the 150-250 nm region.
n * and * transitions: most common transitions observed
in organic molecular UV-Vis, observed in compounds with lone
pairs and multiple bonds with max = 200-600 nm.

Figure from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm

Molecular UV-Vis Spectroscopy: Quantum

Theory

UV-Visible spectra and the states involved in electronic transitions

can be calculated with theories ranging from Huckel to ab initio/DFT.
Example: * transitions responsible for ethylene UV absorption
at ~170 nm calculated with ZINDO semi-empirical excited-states
methods (Gaussian 03W):
HOMO u bonding molecular orbital

LUMO g antibonding molecular orbital

UV Spectroscopy
I.

Introduction
C. Observed electronic transitions
7. From the molecular orbital diagram, there are several possible
electronic transitions that can occur, each of a different relative
energy:

Energy

alkanes

carbonyls

unsaturated cmpds.

O, N, S, halogens

carbonyls

10

UV Spectroscopy
I.

Introduction
E. Band Structure
1. Unlike IR (or later NMR), where there may be upwards of 5 or
more resolvable peaks from which to elucidate structural
information, UV tends to give wide, overlapping bands
2.

It would seem that since the electronic energy levels of a pure

sample of molecules would be quantized, fine, discrete bands
would be observed for atomic spectra, this is the case

3.

In molecules, when a bulk sample of molecules is observed, not

all bonds (read pairs of electrons) are in the same vibrational
or rotational energy states

4.

This effect will impact the wavelength at which a transition is

observed very similar to the effect of H-bonding on the O-H
vibrational energy levels in neat samples

11

UV Spectroscopy
I.

Introduction
E. Band Structure
5. When these energy levels are superimposed, the effect can be
readily explained any transition has the possibility of being
observed
Disassociation
R1 - Rn

V4
R1 - Rn

V3

R1 - Rn

V2
V1 R1 - Rn

E1

Vo

R1 - Rn

Disassociation

Energy

R1 - Rn

V4
R1 - Rn

V3

R1 - Rn

E0

V2
V1 R1 - Rn

Vo

R1 - Rn

12

13

14

UV Spectroscopy
II.

Instrumentation and Spectra

C. The Spectrum
1. The x-axis of the spectrum is in wavelength; 200-350 nm for UV,
200-700 for UV-VIS determinations
2.

Due to the lack of any fine structure, spectra are rarely shown in
their raw form, rather, the peak maxima are simply reported as
a numerical list of lamba max values or max

NH2

max = 206 nm
252
317
376

15

UV Spectroscopy
II.

Instrumentation and Spectra

C. The Spectrum
1. The y-axis of the spectrum is in absorbance, A
2.

From the spectrometers point of view, absorbance is the inverse

of transmittance: A = log10 (I0/I)

3.

From an experimental point of view, three other considerations

must be made:
i. a longer path length, l through the sample will cause
more UV light to be absorbed linear effect
ii.

the greater the concentration, c of the sample, the

more UV light will be absorbed linear effect

iii. some electronic transitions are more effective at the

absorption of photon than others molar absorptivity,

this may vary by orders of magnitude

16

Beers Law Limitation

Polychromatic Light
More than one
wavelength

UV Spectroscopy
IV. Structure Determination
A. Dienes
2. Woodward-Fieser Rules - Dienes
For example:
Isoprene - acyclic butadiene = 217 nm
one alkyl subs.
+ 5 nm
222 nm
Experimental value 220 nm

Allylidenecyclohexane
- acyclic butadiene = 217 nm
one exocyclic C=C + 5 nm
2 alkyl subs.
+10 nm
232 nm
Experimental value 237 nm

18

UV Spectroscopy
III. Chromophores
C. Substituent Effects
1. Conjugation most efficient means of bringing about a
bathochromic and hyperchromic shift of an unsaturated
chromophore:
nm

H2C

max

CH2

175 15,000
217 21,000
258 35,000

465 125,000
-carotene
O

n * 280
* 189

12
900

n * 280
* 213

27
7,100

19

UV Spectroscopy
IV. Structure Determination
C. Aromatic Compounds
2. Substituent Effects
d. Polynuclear aromatics

When the number of fused aromatic rings increases,

the for the primary and secondary bands also
increase

For heteroaromatic systems spectra become complex

with the addition of the n * transition and ring size
effects and are unique to each case
20

UV Spectroscopy
IV. Structure Determination
A. Dienes
3. Woodward-Fieser Rules Cyclic Dienes
There are two major types of cyclic dienes, with two different
base values
Heteroannular (transoid):

= 5,000 15,000
base max = 214

Homoannular (cisoid):

= 12,000-28,000
base max = 253

The increment table is the same as for acyclic butadienes with a

couple additions:
Group
Increment
Additional
homoannular
Where both types of
diene are present, the
one with the longer
becomes the base

+39

21

UV Spectroscopy
IV. Structure Determination
A. Dienes
3. Woodward-Fieser Rules Cyclic Dienes
There are two major types of cyclic dienes, with two different
base values
Heteroannular (transoid):

= 5,000 15,000
base max = 214

Homoannular (cisoid):

= 12,000-28,000
base max = 253

The increment table is the same as for acyclic butadienes with a

couple additions:
Group
Increment
Additional
homoannular
Where both types of
diene are present, the
one with the longer
becomes the base

+39

22

Similar for Enones

X=H207

227

202
215
BaseValues,addtheseincrements

239

ExtndC=C

+30

AddexocyclicC=C

+5

Homoannulardiene

+39

alkyl

+10

+12

OH

+35

+30

EtOH0

OAcyl

+6

+6

+6

+6

CHCl31

Oalkyl

+35

+30

+17

+31

Dioxane5

NR2

X=R215
X=OH193
X=OR193

Withsolventcorrection
of..
Water+8

Et2O7
Hydrcrbn11

Salkyl

+18

+18
+50

23

UV Spectroscopy
IV. Structure Determination
A. Dienes
3. Woodward-Fieser Rules Cyclic Dienes
In the pre-NMR era of organic spectral determination, the power
of the method for discerning isomers is readily apparent
Consider abietic vs. levopimaric acid:

C OH
O

abietic acid

C OH
O

levopimaric acid

24

UV Spectroscopy
IV. Structure Determination
A. Dienes
3. Woodward-Fieser Rules Cyclic Dienes
heteroannular diene =

214 nm

4 alkyl subs. (4 x 5) +20 nm

1 exo C=C
+ 5 nm
C OH
O

239
nm

homoannular diene = 253 nm

4 alkyl subs. (4 x 5) +20 nm
1 exo C=C
+ 5 nm
278
C OH
O

nm

25

UV Spectroscopy
IV. Structure Determination
B. Enones
1. General Features
For auxochromic substitution on the carbonyl, pronounced
hypsochromic shifts are observed for the n * transition (max):
O
H

293 nm

O
CH3

279

Cl

235

NH2

214

204

O
OH

204

This is explained by the inductive

withdrawal of electrons by O, N or
halogen from the carbonyl carbon this
causes the n-electrons on the carbonyl
oxygen to be held more firmly
It is important to note this is different
from the auxochromic effect on *
which extends conjugation and causes
a bathochromic shift
In most cases, this bathochromic shift is
not enough to bring the *
transition into the observed range
26

UV Spectroscopy
IV. Structure Determination
C. Aromatic Compounds
2. Substituent Effects
c. Electron-donating and electron-withdrawing effects

Electron withdrawing

Electron donating

Primary

Secondary

Substituent

max

max

-H

203.5

7,400

254

204

-CH3

207

7,000

261

225

-Cl

210

7,400

264

190

-Br

210

7,900

261

192

-OH

211

6,200

270

1,450

-OCH3

217

6,400

269

1,480

-NH2

230

8,600

280

1,430

-CN

224

13,000

271

1,000

C(O)OH

230

11,600

273

970

-C(O)H

250

11,400

-C(O)CH3

224

9,800

-NO2

269

7,800

27

UV Spectroscopy
III. Chromophores
C. Substituent Effects
General Substituents may have any of four effects on a
chromophore
i. Bathochromic shift (red shift) a shift to longer ; lower
energy
ii.

Hypsochromic shift (blue shift) shift to shorter ; higher

energy

Hyperchromic

iii. Hyperchromic effect an increase in intensity

iv. Hypochromic effect a decrease in intensity

Bathochromic

Hypochromic

200 nm

Hypsochromic

700 nm
28

UV Spectroscopy
V.

Visible Spectroscopy
A. Color
1. General

The portion of the EM spectrum from 400-800 is observable

to humans- we (and some other mammals) have the
adaptation of seeing color at the expense of greater detail

400

500

600

700

, nm
Violet

400-420

Indigo

420-440

Blue

440-490

Green

490-570

Yellow

570-585

Orange

585-620

Red

620-780

29

800

UV Spectroscopy
V.

Visible Spectroscopy
A. Color
1. General

When white (continuum of ) light passes through, or is

reflected by a surface, those ls that are absorbed are
removed from the transmitted or reflected light
respectively

What is seen is the complimentary colors (those that are

not absorbed)

This is the origin of the color wheel

30

UV Spectroscopy
V.

Visible Spectroscopy
A. Color
1. General

Organic compounds that are colored are typically those

with extensively conjugated systems (typically more than
five)

Consider -carotene

-carotene, max = 455 nm

max is at 455 in the far blue region of

the spectrum this is absorbed
The remaining light has the
complementary color of orange

31

UV Spectroscopy
V.

Visible Spectroscopy
A. Color
1. General

Likewise:

lycopene, max = 474 nm

O
H
N
N
H
O

indigo

max for lycopene is at 474 in the near blue region

of the spectrum this is absorbed, the compliment
is now red
max for indigo is at 602 in the orange region of the
spectrum this is absorbed, the compliment is now
indigo!
32

The colors of M&Ms

Bright Blue

Royal Blue

Common Food Uses

Beverages, dairy products, powders, jellies,
confections, condiments, icing.

Common Food Uses

Baked goods, cereals, snack foods, ice-cream,
confections, cherries.

Orange-red

Lemon-yellow

Common Food Uses

Gelatins, puddings, dairy products, confections,
beverages, condiments.

Common Food Uses

Custards, beverages, ice-cream, confections,
preserves, cereals.

Orange
Common Food Uses
Cereals, baked goods, snack foods, ice-cream,
beverages, dessert powders, confections

33

UV Spectroscopy
V.

Visible Spectroscopy
A. Color
1. General

In the chemical sciences these are the acid-base indicators

used for the various pH ranges:

Remember the effects of pH on aromatic substituents

Methyl Orange

O3S

N N

Yellow, pH > 4.4

CH3
N
CH3

O3S

H
N N

Red, pH < 3.2

34

CH3
N
CH3

How Do UV spectrometers
work?
Rotates,to
achievescan

Matchedquartzcuvettes
Sampleinsolutionatca.105M.
SystemprotectsPMtubefrom
straylight
D2lampUV
TungstenlampVis
DoubleBeammakesita
differencetechnique
Twophotomultiplier
inputs,differential
voltagedrivesamplifier.

35

Diode Array Detectors

Diodearray
alternativeputs
grating,arrayof
photosens.
Semiconductorsafter
thelightgoesthrough
thesample.
Advantage,speed,
sensitivity,
TheMultiplex
advantage

ModelfromAgilentliterature.Imagine
replacingcellwithamicroflowcellfor
HPLC!

Disadvantage,
resolutionis1nm,vs
0.1nmfornormal
UV
36

Solvents for UV (showing high

energy cutoffs)
Water

205

THF

220

CH3CN 210

CH2Cl2 235

C6H12

210

CHCl3

245

Ether

210

CCl4

265

EtOH

210

benzene

280

Hexane

210

Acetone

300

MeOH

210

Dioxane

220

Variousbuffersfor
HPLC,checkbefore
using.

37

Quantitative
analysis
Greatfornon
aqueoustitrations
Examplehere
givesdetnof
endpointfor
bromcresolgreen

Isosbesticpoints
Singleclearpoint,canexclude
intermediatestate,excludelight
scatteringandBeerslawapplies

Bindingstudies
FormItoformII

Bindingofalanthanidecomplexto
anoligonucleotide

38

Isosbestic point

-At different pH, the spectrum will be shifted to different max

but all spectra intersect at certain which is known as isosbestic
point
- At isosbestic point, the same absorbance is given for the same

concentration at different pH,i.e. absorbance is not pH

dependent but concentration dependent
-Thus solution ; its max affected by pH , must be buffered at
specific pH or measurements are carried out at the isosbestic
39
point.

More Complex Electronic Processes

Fluorescence: absorption of
radiation to an excited state,
followed by emission of radiation
to a lower state of the same
multiplicity
Phosphorescence: absorption of
radiation to an excited state,
followed by emission of radiation
to a lower state of different
multiplicity
Singlet state: spins are paired, no
net angular momentum (and no
net magnetic field)
Triplet state: spins are unpaired,
net angular momentum (and net
magnetic field)

40

Parameters
Extinction Coefficient

refers to a single wavelength (usually the absorption

maximum) the cross sectional area of a molecule determines
how efficient it will absorb photons

Quantum Yield
Qf

is a measure of the integrated photon emission over the

fluorophore spectral band

At sub-saturation excitation rates,

fluorescence intensity is proportional to
the product of and Qf

41

Fluorescence
Quantum Yield

Q=

photons emitted
=
photons absorbed

kr
kr + knr

Fluorescence Lifetime (
- is the time delay between the absorbance and
the emission

1
kr + knr
R - reactant molecules)

42

Fluorescence
Photon emission as an electron
returns from an excited state to
ground state

43

Fluorescence
Excitation Spectrum
Intensity of emission as a function of
exciting wavelength (this is the
absorbance component)

Chromophores are components of

molecules which absorb light
They are frequently aromatic rings

44

Fluorescence
The wavelength of absorption is
related to the size of the
chromophores
Smaller chromophores, higher energy
(shorter wavelength)

45

Properties of Fluorescent Molecules

Large extinction coefficient at the

region of excitation
High quantum yield
Optimal excitation wavelength
Photostability
Excited-state lifetime
Minimal perturbation by probe

46

Simplified Jablonski Diagram

S
1

Energy

S1

hvex

hvem

S0

Slide 47

Fluorescence
Jablonski Diagram
Singlet States

Triplet States
Vibrational energy levels
Rotational energy levels
Electronic energy levels

ENERGY

S2

S1

IsC

ABS

FL

I.C.

T2
T1

PH
IsC

S0

[Vibrational sublevels]

ABS - Absorbance
S 0.1.2 - Singlet Electronic Energy Levels
FL - Fluorescence
T 1,2 - Corresponding Triplet States
I.C.- Nonradiative Internal Conversion IsC
- Intersystem Crossing PH - Phosphorescence

3rd Ed. Shapiro p 87

4th Ed. Shapiro p 112

48

Fluorescence
Stokes Shift

Fluorescnece Intensity

is the energy difference between the

lowest energy peak of absorbance
and the highest energy of emission
Fluorescein
molecule

Stokes Shift is 20 + nm
495 nm

Wavelength

518 nm

49

Fluorescence
The longer the wavelength the lower
the energy
The shorter the wavelength the
higher the energy
eg. UV light from sun - this causes the
sunburn, not the red visible light

50

Fluorescence Excitation Spectra

Intensity
related to the probability of the event

Wavelength
the energy of the light absorbed or emitted

51

Conclusions
Dye molecules must be close to but
below saturation levels for optimum
emission
Fluorescence emission is longer than
the exciting wavelength
The energy of the light increases with
reduction of wavelength

52

Some Common Laser lines

350
300 nm

457 488 514

400 nm

500 nm

610 632
600 nm

700 nm

Common Laser
Lines

PE-TR Conj.
Texas Red
PI
Ethidium
PE
FITC
325nm

cis-Parinaric acid
53

Quenching, Bleaching & Saturation

Quenching is when excited molecules relax to

ground states via nonradiative pathways
avoiding fluorescence emission (vibration,
collision, intersystem crossing)
Molecular oxygen quenches by increasing the
probability of intersystem crossing
Polar solvents such as water generally quench
fluorescence by orienting around the exited
state dipoles
3rd Ed. Shapiro p 90
4th Ed. Shapiro p 115

54

Photobleaching
Defined as the irreversible destruction of
an excited fluorophore
Photobleaching is not a big problem for
flow cytometry as long as the time
window for excitation is very short (a few
hundred microsconds)

55

Photobleaching example from microscopy

FITC - at 4.4 x 1023 photons cm-2 sec-1 FITC

bleaches with a quantum efficiency Qb of 3
x 10-5
Therefore FITC would be bleaching with a
rate constant of 4.2 x 103 sec-1 so 37% of
the molecules would remain after 240 sec
of irradiation.
In a single plane, 16 scans would cause 650% bleaching

56

Excitation Saturation

The rate of emission is dependent upon the time the

molecule remains within the excitation state (the excited
state lifetime f)

Optical saturation occurs when the rate of excitation

exceeds the reciprocal of f

In a scanned image of 512 x 768 pixels (400,000 pixels) if

scanned in 1 second requires a dwell time per pixel of 2 x
10-6 sec.
Molecules that remain in the excitation beam for extended
periods have higher probability of interstate crossings and
thus phosphorescence
Usually, increasing dye concentration can be the most
effective means of increasing signal when energy is not the
limiting factor (i.e. laser based confocal systems)

57

Phosphorescence
Following absorption, molecules can relax via a
non-radiative transition to the T1 rather than the S1
state - this is called an intersystem crossing
While it is forbidden it does happen and has a low
probability and takes a longer time - the energy
dissipated is called phosphorescence
Phosphorescence has a longer lifetime than
fluorescence (milliseconds rather than
femtoseconds
Phosphorescence generally occurs at longer
wavelengths than fluorescence because the
energy difference between S0 and T1 is lower
3rd Ed Shapiro p 88
4th Ed. Shapiro p 113
58

Excitation - Emission Peaks

Fluorophore

EXpeak EM peak

FITC
496 518 87
Bodipy
503 511
Tetra-M-Rho
554 576
L-Rhodamine
572 590
Texas Red
592 610 3
CY5
649 666 1

0
58
10
5
45
11

% Max Excitation at
488 568 647 nm

0
1 1
61 0
92 0
1
98

Note: You will not be able to see CY5 fluorescence

under the regular fluorescent microscope because
the wavelength is too high.

Slide 59

Resonance Energy
Transfer
Resonance energy transfer can occur when the
donor and acceptor molecules are less than
100 of one another (preferable 20-50 )
Energy transfer is non-radiative which means
the donor is not emitting a photon which is
absorbed by the acceptor
Fluorescence RET (FRET) can be used to
spectrally shift the fluorescence emission of a
molecular combination.
3rd Ed. Shapiro p 90
4th Ed. Shapiro p 115
60

Fluorescence Resonance Energy Transfer

Molecule 1

Molecule 2
Fluorescence

Intensity

Fluorescence
ACCEPTOR

DONOR

Absorbance
Absorbance

Wavelength
Slide 61

Fluorescence
The longer the wavelength the lower the energy
The shorter the wavelength the higher the energy
eg. UV light from sun - this causes the sunburn, not the red visible light

The spectrum is independent of precise excitation line but the

intensity of emission is not

Slide 62

Fluorescence Instrumentation
Xenon Source

Excitation
Monochromator

Emission
Monochromator

PMT

Sample compartment
63

Lecture Summary

Light and Matter

UV-Vis Absorption
Fluorescence

From this lecture you should understand:

The nature of UV active molecules

The relationship of concentration to light adsorption
The nature of fluorescence molecules
How fluorescence is generated
Why molecules have different excitation and emission
What Resonance Energy Transfer is

Slide 64