Ultraviolet –

Visible Spectroscopy
Electromagnetic radiation spectrum

visible (VIS) regions 400-750 nm

ultraviolet (UV) region, 100-350 nm

The Spectrum and Molecular Effects

Energy per photon = hν, where h is Planck’s constant.

 Interaction of electromagnetic radiation with
molecules

In order to excite an
E4
atom/molecule from E1 to E2
it must absorb an amount of E3
energy equivalent to A12
E2 Excited states
Energy
A12 E21 E = h
h

E1
Ground state

Discrete Energy levels of an atom or molecule

 The energy of electronic excitation

The energy is related to wavelength by the equation:

E = h = hc/

Where h is the Plank’s constant and c is the speed of light.

The energy absorbed is dependent on the energy difference between the

ground state and the excited state.

The smaller the difference the longer the wavelength of absorption.

 The UV-visible Spectroscopic Process
1. In UV spectroscopy, the sample is irradiated with the broad spectrum of
the UV-visible radiation
2. If a particular electronic transition matches the energy of a certain band
of UV, it will be absorbed
3. The remaining UV light passes through the sample
4. A spectrum is obtained – which is called an absorption spectrum




UV radiation 


An Electronic Spectrum

1.0
maxwith certain
extinction  UV Visible
Absorbance

0.0
200 400 800
Wavelength, , generally in nanometers (nm)
 Instrumentation and Spectra

Simple schematic that covers most modern UV spectrometers

log(I0/I) = A
UV-VIS sources I0 I

sample
200

detector
, nm

monochromator/ 700
beam splitter optics I0 reference I0
 Basic components of Instrument

 Two sources are required to scan the entire UV-VIS band:

 Deuterium lamp – covers the UV – 200-350
 Tungsten lamp – covers 350-700

 The lamps illuminate the entire band of UV or visible light; the

monochromator (grating or prism) separates the different
wavelengths of light and sends them to the beam splitter.

 The beam splitter sends a separate band to a cell containing the

sample solution and a reference solution

 The detector measures the difference between the transmitted

light through the sample (I) vs. the incident light (I0) and sends
this information to the recorder
 Instrumentation
• Light source
• Deuterium and hydrogen lamps (160-375 nm)
Emit light by electrical excitation of deuterium
• Tungesten filament lamp (common source of visible and
near IR light)
• Xe arc lamps
• Sample containers
• Cuvettes A typical sample cell
(commonly called a
• Plastic
• Glass
cuvett)
• Quartz
• Acrylic-visible, UV
Only quartz is transparent in the
Plastic and glass are only
full 200-700 nm range
suitable for visible spectra

 Comes with different

path length!
Sample Preparation

• A dilute sample and a reference sample containing pure

solvent is prepared.

• Cells are placed in the appropriate place in the spectrometer.

• Two equal beams of light are passed, one through the

solution of the sample, one through the pure solvent.

• The intensities of the transmitted light are measured.

• The spectrum is plotted as Absorbance (A) or transmittance

vs wavelength. The λ unit is almost always in nanometers
(nm).
Choice of Solvent
Solvents must be transparent; the wavelength where a
solvent is no longer transparent is referred to as the cutoff

These are the minimum wavelength (for a 1 cm cell).

Common solvents and

cutoffs:
acetonitrile
190
chloroform
240
cyclohexane
195
1,4-dioxane
215
95% ethanol
205
Transparency ranges for common solvents in
UV-visible spectroscopy

Quartz and
glass are used
to make the
sample cells
for electronic
spectra. The
fig shows why
glass can not
be used for UV
cells.
 Laws of Absorbance
The intensity of light decreases when it passes through an absorbing
medium
Absorption, A = log (I0/I) I0 I

sample
Or
The intensity of the absorption band is measured by the percent of the
incident light that passes through the sample:

% Transmittance = (I / I0) * 100%

where:

I = intensity of transmitted light

I0 = intensity of incident light

A ( absorbance) will depend on three factors

i. path length, l: is the length through which the light is

passing (Cuvvet dimensions)

ii. Concentration, c of the sample

iii. molar absorptivity, (epsilon) of the sample- determines the

number and type of chromophores present in each molecule
(characteristic of the substance) (is a measure of how well a
given material absorbs light)
 Beer-Lambert Law
These effects are combined into the Beer-Lambert Law:
A=cl

where: ε = molar absorptivity (L mol-1 cm-1)

c = molar concentration of
solute (moles/Lt)
l = length of sample cell (cm)

l is constant (standard cells are typically 1 cm in path length)

Concentration is typically varied depending on the strength of absorption

observed or expected – typically dilute – sub .001 M

Molar absorptivities vary by orders of magnitude:

•values of 104-106 are termed high intensity absorptions
•values of 103-104 are termed low intensity absorptions
•values of 0 to 103 are the absorptions of forbidden transitions
Absorbance is directly related to concentration (for dilute
solution) ~ in the order of x10-5 M
 Limits to Beer’s Law
Causes to deviations
• Fails at high concentrations (molecules influence each other)

• If concentration changes, this can also cause changes in

refractive index resulting in deviation.

• Reflections and scattering

 Limits to Beer’s Law

Polychromatic radiation (i.e. Beer’s law only true for

monochromatic light.

Stray radiation (changes in wavelength) can cause changes to

calibration curve.

 includes leakage radiation, residual

radiation, and scattered radiation
from irradiated objects.
 UV-Vis spectrum of 4-
nitroaniline in EtOH Interpretation of a UV spectrum
recorded 15.4 mg L-1, path
length = 1cm.
Determine  values for the two crests seen in the
spectrum at max shown.

max = 227 nm, A = 1.55, max = 375 nm, A = 1.75

The molecular weight of 4-nitroaniline = 138 g/mol

Concentration of the solution = 15.4 mg/L
= 154 x 10-4 g/L
Molarity of the solution = (154 / 138) x 10-4 mol/L
= 1.12 x 10-4 mol/L

As absorbance (A) = cl;  = A/cl and as l = 1 cm,

 = A/c

Absorption onset Therefore: 227nm) = 1.55/1.12 x 10-4 = 13890

Similarly; 375nm) = 1.75/1.12 x 10-4 = 15680

Quote these results as max (EtOH) 227 ( 13890), 375 (

15680)
 Observed electronic transitions and Absorbing species

• Electronic transitions
 and n electrons (non-
bonding)
 d and f electrons
Here is a graphical representation
 Charge transfer reactions

Unoccupied levels


Atomic orbital Atomic orbital

Energy n
For every bond
there are two
contributing
atomic orbital Occupied levels



Molecular orbitals
 Electron transitions
From the molecular orbital diagram, there are several possible electronic
transitions that can occur, each of a different relative energy

  Alkanes (150 nm)

  unsaturated cmpds.
(180nm)

n  O, N, S, halogens
(190nm, observable
when conjugated)

n  Carbonyls (300nm,
Observable but weak
Peak)
 Chromophores
1.A functional group capable of having characteristic electronic
transitions is called a chromophore (color loving)

2.Structural or electronic changes in the chromophore can be

quantified and used to predict shifts in the observed electronic
transitions
 Organic Chromophores
Alkanes – only posses -bonds and no lone pairs of electrons, so only the
high energy   * transition is observed in the far UV

This transition is destructive to the molecule, causing cleavage of the -

bond

Alcohols, ethers, amines and sulfur compounds –

n  * is the most often observed transition; like the alkane   * it is most
often at shorter  than 200 nm

Alkenes and Alkynes –

 * is observed at 175 and 170 nm, respectively
Even though this transition is of lower energy than   *, it is still in the
far UV region

Carbonyls –
normally observes n  * transitions (~285 nm) in addition to    *

This transition is forbidden by the selection rules ( = 15), but is often

observed for carbonyls
 Substituent Effects
In 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

iii. Hyperchromic effect – an increase in intensity

iv. Hypochromic effect – a decrease in intensity

Hyperchromic


Hypsochromic Bathochromic
Hypochromic

200 nm 700 nm
Substituents that increase the intensity and often wavelength of an absorption are
called auxochromes

The attachment of substituent groups (other than H) can shift the energy of the
transition

Common auxochromes include alkyl, hydroxyl, alkoxy and amino groups and the
halogens

For example in an alkene:

Methyl groups can cause a bathochromic
shift, even though they are devoid of - or H

n-electrons C
This effect is thought to be through what is C C H
H

termed “hyperconjugation” or sigma bond

resonance
 Effects - Conjugation – Alkenes

The molecular orbital of ethene and butadiene - note how conjugation reduce the
HOMO-LUMO energy difference.

E for the HOMO  LUMO transition is reduced-shifts the absorption to the red
Extending this effect out to longer conjugated systems the energy gap
becomes progressively smaller:

Energy Lower energy =

Longer wavelengths

ethylene
butadiene
hexatriene
octatetraene
Conjugation – most efficient means of bringing about a bathochromic and
hyperchromic shift of an unsaturated chromophore:

H2C
max nm 
CH2
190 15,000

217 21,000

258 35,000

465 125,000

-carotene

O
n  * 280 12
  * 189 900
O n  * 280 27
  * 213 7,100
Effect of conjugation on optical absorption spectrum

MeO OMe
MeO OMe

S
N N N N
O O
Zn 3
Br Br Br Br Monomer
Acetic acid Acetic acid
O2N NO2 N N
21

MeO OMe
DA-267

Conjugated Polymer
 Effects of PH upon the absorption maxima of
phenols
OH O O O OH

Base resonance

(e.g. -OH

Phenol Phenoxide
anion

Neutral or acidic conditions Basic conditions

max (H2O) 210 (6000) max (H2O) 235 (9400)
270 (1500) 287 (2600)

Phenols are acidic, therefore the addition of base increases the conjugation
of the lone pairs on the oxygen with the -system of the aromatic ring.

This leads to a decrease in the energy difference between the HOMO and the
LUMO orbitals, resulting in a red or bathochromic shift, along with an
increase in the intensity of the absorption.
 The UV-Vis spectra Absorption onset
~290 nm
of phenol

Absorpti
on onset
(~300
nm)

The UV-Vis spectra of phenol (in ethanol, black)

and the phenolate ion (in 0.1 M NaOH, red)
 Effects of Ph upon the absorption maxima of
an aromatic amine

NH2 NH2 NH3

Resonance acid (e.g H+)

Aniline Protonated
Neutral or basic conditions Acid conditions
max (H2O) 235 (148000) max (H2O) 203 (7500)
285 (2800) 254 (180)

Aromatic amines are basic, therefore, the treatment of amines with acids, on
the other hand, causes protonation of the amine and this leads to a loss of
the overlap between amine lone pairs and the aromatic -system of the
aromatic ring.

This results in a blue or hypsochromic shift, along with an decrease in the

intensity of the absorption.
 UV-Vis spectra of aniline

UV-Vis spectra of aniline (in ethanol, black)

and the anilinium ion (in 0.1 M HCI, red)

Useful in pH indicators
 Effects of solvent polarity
The solvent in which the UV spectrum is measured can have an effect on
the position of max.

The Frank-Condon principle states that, during electronic excitation,

atoms do not move, but electrons -including those of the solvent-may
reorganize themselves.

If the solvent being used is polar, it can stabilize the excited state more
readily by dipole-dipole interactions than the ground state.

The bathochromic effect in

the UV spectra of 4-
nitroaniline recorded in
hexane and ethanol.