UV-Visible Molecular

Absorption Spectrometry
Introduction and Background

• Involves absorption of ultraviolet,

visible, or infrared radiation for
quantitative purposes.

• Most common analytical technique in

the analytical laboratory

• Absorption commonly occurs with many

– Organic molecules
– Metals
– Metal-organic complexes
 Questions

• What is nature of light?

• Are there different types of

light?

• How does light propagate?

 What is Light?
• Light is a form of energy
• Light travels through space at extremely
high velocities
– The speed of light (c) ~ 3 x 1010 cm/sec or
186,000 miles per second
• Light is classified as electromagnetic
radiation (EMR)
Characteristics of Light
• Light behaves like a wave.
– That is, it can be modeled or characterized with
wave like properties.

• Light also behaves like a particle.

• Today, we envision light as a self-

contained packet of energy, a photon,
which has both wave and particle like
properties.
The Electromagnetic Spectrum
The Electromagnetic Spectrum
The EMR
Spectrum
Different portions of
the EMR spectrum and
different types of
spectroscopy involve
different parts
(quantum states) of the
atom
 EMR Wave Characteristics
• Wavelength (λ ) is the distance from one wave crest to the
next.
• Amplitude is the vertical distance from the midline of a wave
to the peak or trough.
• Frequency (v) is the number of waves that pass through a
particular point in 1 second (Hz = 1 cycle/s)
 Wave Properties of
Electromagnetic Radiation

• EMR has both electric (E) and magnetic (H) components that
propagate at right angles to each other.
 Particle Properties of EMR

• The energy of a photon depends on

its frequency (v)

Ephoton = hv

h = Planck’s constant
h = 6.63 x 10-27 erg sec or 6.63 x 10-34 Js
 Electromagnetic Radiation

V = Wave Number (cm ) -1

λ = Wave Length
C = Velocity of Radiation (constant) = 3 x 1010 cm/sec.
υ = Frequency of Radiation (cycles/sec)
υ 1
V = =
C λ
The energy of photon:
h (Planck's constant) = 6.62 x 10-27 (Erg× sec)

C C
E = hυ
=h υ= C=
λ λ
υ λ
How Light Interacts with Matter.

• Atoms are the basic blocks

of matter.
• They consist of heavy
particles (called protons
and neutrons) in the
nucleus, surrounded by
lighter particles called
electrons
 How Light Interacts with Matter.

• An electron will interact with a photon.

• An electron that absorbs a photon will gain
energy.
• An electron that loses energy must emit a
photon.
• The total energy (electron plus photon) remains
constant during this process.
How Light Interacts with Matter.
• Electrons bound to
atoms have discrete
energies (i.e. not all
energies are allowed).
• Thus, only photons of
certain energy can
interact with the
electrons in a given
atom.
How Light Interacts with Matter.
• Consider hydrogen, the
simplest atom.
• Hydrogen has a specific line
spectrum.
• Each atom has its own specific
line spectrum (atomic
fingerprint).
 Unique Atomic Signatures
Each atom has a specific set of energy levels, and thus a
unique set of photon wavelengths with which it can interact.
 Molecular Absorption
• More complex than atomic absorption
because many more potential transitions
exist
– Electronic energy levels
– Vibrational energy levels
– Rotational energy levels
• Emolecule = Eelectronic + Evibrational + Erotational
– Eelectronic > Evibrational > Erotational
• Result - complex spectra
Energy Level Diagram for
Molecular Absorption
Molecular Absorption Spectra
of Benzene in the Gas Phase
Electronic Transition
Vibrational Transition
Superimposed on the
Electronic Transition

Absorption Band –
A series of closely
shaped peaks
 Molecular
Absorption
Spectra in the
Solution Phase

• In solvents the
rotational and
vibrational transitions
are highly restricted
resulting in broad band
absorption spectra
 Emission of EMR
• EMR is released when excited atoms or
molecules return to ground state
– Reverse of the absorption process
– We call this process “emission”
• Initial excitation can occur through a number
of pathways
– Absorption of EMR
– Electrical discharge
– High temperatures (flame or arc)
– Electron bombardment
 Emission of EMR
• We distinguish several types of emission
1. Atomic
2. X-Ray
3. Fluorescence
Involves molecules
Resonance and non-resonance modes
1. Phosphorescence
• Non-radiative relaxation
• Similar to fluorescence only relaxation times are
slower than fluorescence
• Involves metastable intermediates
Energy Level Diagrams of
Excitation and Emission
 Absorption by
Organic Compounds

Many common organic

compounds absorb in
the UV region
 Absorption by
Inorganic Species

Many free metals and

inorganic metal
complexes absorb in
the visible region of
the spectrum
 A few metal chlorides,
which fluoresce strongly in
the visible wavelengths,
are the basis for almost
all the colors in modern fireworks.

Barium chloride produces green;

strontium chloride produces red;
copper chloride produces blue
Absorption by Charge
Transfer Complexes
• Many inorganic and organic
complexes form charge
transfer complexes
• A charge transfer complex
consists of an electron donor
group bonded to an electron
acceptor group
• Charge transfer complexes
exhibit broad band
absorption in the visible
region of the EMR spectrum
• Useful analytically because of
the large molar absorption
Charge-Transfer Complex
Charge-Transfer Complex
• Nitrite can be
λmax determined analytically
by adding reagents to
form a colored charge
transfer complex.

• The complex exhibits

broad band absorption
in the visible region of
the EMR spectrum

• The wavelength of
maximum absorption
(λmax) can be determined
with a wavelength scan

• Measurements are then

made at λmax
 Choice of Solvents
• Most absorption measurements are
conducted by dissolving the analyte in a
solvent
• The solvent (and sample holder) should
be transparent in the region of the
spectrum where the analyte absorbs
Single Beam Instruments
Double-Beam Instruments
• A double beam instrument is one in
which the light source can be passed
(simultaneously) through both a
reference and a sample cell

• Purpose and Approach

1. Adjust light output of the instrument
to 100% transmission (0 %
absorbance)
2. Allows correction of the sample
absorbance signal for non-analyte
absorbance
 Double-Beam Instruments
Reference and Sample Cell Options
Reference Reference Sample or Standard
Cell Cell Cell
Solution (pure H2O) Solution (pure H2O) Solution (pure H2O)
Reagents Reagents
Analyte or Sample

Signal Due to Signal Due to

Reagents Only Analyte Only
(Can be used to
estimate
reagent blank)
 Dispersion of Polymagnetic Light with a Prism

Prism - Spray out the spectrum and choose the certain wavelength
(λ ) that you want by slit.

Infrared
monochromatic
Ray

Red
Orange
Yellow SLIT
Polychromatic PRISM
Green
Ray Blue
Violet

Ultraviolet

Polychromatic Ray Monochromatic Ray

Double-Beam Instruments
• Example component layout for a double
beam instrument
• Light beam is split using a “chopper”
Double Beam Instruments
Mechanical Chopper
• The rotating disk blocks
the transmission of light
(% T = 0)
• Putting a mirror on the
face of the rotating disk
will re-direct the light to
an alternate path
Double-Beam Instruments
Used to provide a
reference and sample
path for the source
light

Used to correct for

non-analyte
absorption signals :
-Reflected or stray
light
-Analyte in the
reagents
-Absorption by
reagents
 Example of UV-
Visible Instrument
 Ultra Violet Spectrometry

The absorption of ultraviolet radiation by molecules is

dependent upon the electronic structure of the molecule.
So the ultraviolet spectrum is called electronic spectrum.
 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.
 Antibonding
σ*

π
* Antibonding
σ

π
n→
σ
σ→

π
*

*
n→*
*
Energy

n Nonbonding
Bonding
π

Bonding
σ
 Electronic Molecular Energy Levels

The higher energy transitions (σ →σ *) occur a shorter

wavelength and the low energy transitions (π →π *, n
→π *) occur at longer wavelength.
Chromophore is a functional group which absorbs a
characteristic ultraviolet or visible region.
UV
210 nm Double Bonds
233 nm Conjugated Diene
268 nm Conjugated Triene
315 nm Conjugated Tetraene

• •
• •

σand σ
* orbitals πandπ
* orbitals
 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
 Spectroscopy Terms Describing
Absorption (Beer’s Law)

• Consider a beam of light

with an (initial) radiant
intensity Po

• The light passes through a P0 hv

P
solution of concentration (c)

• The thickness of the

) c( noi t art nec no C

solution is “b” cm.
b

• The intensity of the light

after passage through the
solution (where absorption
occurs) is P
 We Define

• Transmittance (T) = P/P0 (units = %)

• Absorbance (A) (units = none)
– A = log (P0/P)

– A = -log (T) = log (1/T)

– A = abc (or εbc) <--- Beer’s Law

• a = absorptivity (L/g cm)
• b = path length (cm)
• c = concentration (g/L)
• ε = molar absorptivity (L/mol cm)
– Used when concentration is in molar units
 Beer’s Law
Major Point:
There is a linear relationship between
absorbance and concentration (but not
absorbance and transmission)
A = abc = εbc
= log (Po/P)
= log (1/T)
 Example

P0 = 10,000 P = 5,000

-b-
P 5000
T = = = 0.5
P0 10000

A = -log T = -log (0.5) = 0.3010

 Example

P0 = 10,000 P = 2,500

--2b--
P 2500
T = = = 0.25
P0 10000

A = -log T = -log (0.25) = 0.6021

Absorption vs.
3.5
3.0

Transmission

Absorbance
2.5
2.0
1.5
A = abc
1.0
0.5
0.0
0 1 2 3 4 5 6 7 8
Thickness, multiples of b
1.2
1
T = 10-abc

Transmittance
0.8
0.6
0.4
0.2
0
Absorption vs. Transmission
A similar C alibration curve to calculae concentration of unknow n from
C onc . inM A bsorbanc es
treatment can also 30.00 0.162 0.90
be shown using 60.00 0.330 0.80
0.70
concentration 90.00
120.00
0.499
0.660
0.60

Absorbance
0.50
150.00 0.840 0.40
unknown 0.539 0.30
0.20
Determining R egression equation
0.10
slope 0.00562
Concentrations from Interc ept -0.0076
0.00
30.00 60.00 90.
Absorbance Data – C onc of unknown 97.25978648 C o n c,
E rror Analysis
Calibration Curves sr (standard error in y ) 0.004802777
N 5
Sx x 9000
y bar (average absorbanc e) 0.498
M (number of replic ates) 1
sc 0.938434226
S pr eadsheet D ocumentation
C ell B 10= S LOP E (B 3:B 7,A 3:A 7)
C ell B 11= INTE RC E PT(B 3:B7,A 3:A 7)
 Limitations to Beer’s Law
• Real
– At high concentrations charge distribution effects occur
causing electrostatic interactions between absorbing
species
• Chemical
– Analyte dissociates/associates or reacts with solvent
• Instrumental
– ε = f(λ); most light sources are polychromatic not
monochromatic (small effect)
– Stray light – comes from reflected radiation in the
monochromator reaching the exit slit.
Instrumental Limitations - ε = f(λ)

• How/Why does ε vary

with λ?
• Consider a wavelength
scan for a molecular
compound at two
different wavelength
bands
• In reality, a
monochromator can
not isolate a single
wavelength, but rather
a small wavelength
band
 Instrumental Limitations –
Stray Light

• How does stray light effect Absorbance and

Beer’s Law?
• A = -log P/Po = log Po/P

• When stray light (Ps) is present, the

absorbance observed (Aapparent) is the sum of
the real (Areal) and stray absorbance (Astray)
 Instrumental Limitations –
Stray Light
• Result – non-linear
absorption (Analyte
vs. Conc.) as a
function of analyte
concentration
– Similar to
polychromatic light
limitations
 VITAMINS

Vitamin A

CH3
H3C CH3

CH3 CH3

CH2OH
β - Carotene

CH3
CH 3 CH 3 CH 3
H3 C

CH3
CH 3 CH 3 CH3
CH3

Oxidation

CH 3 CH3 O
H3 C CH3
C H

CH3 Retainal

- 2H

CH 3 CH3
H3 C CH3
CH 2 OH

CH3 Retinol (Vitamin A)

Vitamin A and β - Carotene Determination

Food
KOH (Alcoholic)
Saponification

3 hrs. at room temperature

Ether for Extraction

Extract (vit.A and carotenoids)

Total Carotenoids Vitamin A + Carotenes

only at 440 nm Carr-Price Reagent
Measure at 620 nm
β -CAROTENE STANDARD ABSORBANCES AT 440 AND 620 nm

and 620 nm

A at 440 nm
A at 440 nm

Aat 620 nm
Use absorbances at 440 nm and then convert this to absorbance at
620 nm and subtract from the absorbance at 620 nm to determine
the absorbance at 620 due to Vitamin A.

Carotenoid Absorbance at 440nm Vitamin A Absorbance at 620 nm

x
x
Absorbance at 440nm

Absorbance at 62 nm
x
x

0
x
x
x
x
x x

Carotenoid (µ g/ml) Vitamin A µ g/cuvette (sample)

 THIAMIN DETERMINATION

pyrimidine thiazole
H3C N NH 2 S CH 2CH 2OH

N N
CH 2 CH 3

K3 Fe(CN)6
Oxidation

H3C N N S CH 2CH 2OH

N N
CH 2 CH 3
THIOCHROME (Fluorescent)

Excite thiochrome at 365 nm and measure the absorbance at 435 nm

RIBOFLAVIN DETERMINATION

CH 2OH
HOCH
HOCH
HOCH
HCH
CH 3 N N O
8 9 1
7 2
6 10 3
5 4
CH 3 N
O

6,7 Dimethyl-9-D-1-Ribitylisoalloxazine
 NIACIN

O O
C OH C NH 2

N N
 Niacine Determination

1 O
O
-
C OH C OHREARRANGEMENT
REARRANGED
+ CNBr RING IS OPENED DERIVATIVE
N N
Br CN

2
O
RERRANGED
+ H2 N S OH
DERIVATIVE
O
O
C-OH O
R-N-CH=CH-CH=C-CH=N S OH
O
 NIACINE STANDARD CURVE

A at 470 nm

µg Niacin
 VITAMIN C
Ascorbic Acid Dehydroascorbic Acid

CH2OH CH2OH
HOCH O HOCH O
-2 H
O O
+ 2H

HO OH O O

HSCH2CH2(SH)CH2OH 2,3-DIMETHYLPROPANOL
Reducing agent or converting dehydroascorbic acid to ascorbic acid
 Titrimetric Method for Reduced Vitamin C

1. Extract with metaphosphoric acid (HPO3) in HOAC.

2. Titrate with 2, 6-dichlorophenolindophenol (blue).
At end point, rose pink (2, 6-dichlorophenolindophenol in acidic
condition).