CHAPTER 2A:

MOLECULAR ABSORPTION
SPECTROSCOPY :
THEORY, INSTRUMENTATION &
APPLICATION

Prepared by:
Wan Elina Faradilla Wan Khalid
Faculty of Applied Science
UiTM Negeri Sembilan
1
 1. COMPONENTS OF
INSTRUMENTS FOR
OPTICAL SPECTROSCOPY

2
 Five Basic Optical Instrument
Components

1) Source A stable source of radiant energy.

2) Sample Container A transparent container used to hold

the sample (cells, cuvettes, etc).

3) Wavelength Selector A device that isolates a restricted

region of the spectrum for measurement
(monochromators, prisms & filters).

4) Detector/Transducer Converts radiant energy to a

measureable electrical signal .

5) Signal Processor & Readout Amplifies or attenuates

the transduced signal and sends it to a readout device
(meter, digital readout, chart recorder, computer)
3
 I. Sources of Radiation

Generate a beam of radiation that is stable and has

sufficient power.

A. Continuum Sources emit radiation over a broad

wavelength range and the intensity of the radiation
changes slowly as a function of wavelength.

This type of source is commonly used in UV, visible and

IR instruments.

- Deuterium lamp is the most common UV source. If

need high intensity use high pressure Xe, Hg, or Ar lamp
- Tungsten lamp is the most common visible source.
- Nerst Glower are common sources for IR
instruments.
4
B. Line Sources Emit a limited number lines or
bands ofradiation at specific wavelengths.

- Used in atomic absorption spectroscopy

- Usually provide radiation in the UV and visible

region of the EM spectrum.

- Types of line source:

1) Hollow cathode lamps
2) Electrodeless discharge lamps
3) Lasers-Light
5
 II. Wavelength Selectors

Instruments in UV and visible region usually

equipped with one or more devices to restrict the
radiation being measured to narrow band that is
absorbed or emitted by the analyte.

In addition, for absorption measurements - narrow

bands radiation greatly diminish the chance of
Beers Law deviations due to polychromatic
radiation.

Many instruments use a monochromator or filter

to isolate the desired wavelength band so that
only the band of interest is detected and
measured.
6
 Two types of wavelength selectors:

1) Filters select a specific, narrow

region of the spectrum for
transmission to the detector and rejects
all the wavelength outside this region

2) Monochromators - separate, isolate

and controls the intensity of radiant
energy reaching the detector
Monochromator generally provide more precisely selected wavelength (a narrower range of
wavelength) than filter.
7
 A. Filters

- Two types of filters:

1) Interference Filters UV, VIS, IR
2) Absorption Filters limited to VIS region, less expensive
than interference filter

B. Monochromators

Wavelength selector that can continuously scan a broad range of

wavelengths.

Monochromator are designed for spectral scanning.

Used in most scanning spectrometers including UV, visible, and IR

instruments.

8
 III. Radiation Transducer (Detectors)

Detector: A device that indicates the existence of some physical

phenomenon.

Example: mercury level in thermometer (indicating temperature)

Early detectors in spectroscopic instruments were the human eye,

photographic plates or films. Modern instruments contain devices that
convert the radiation to an electrical signal.

Transducer: type of detector that converts quantity into electrical

signal (pH, mass, temperature, intensity)

Two general types of radiation transducers:

a. Photon detectors respond to photon
b. Thermal detectors respond to heat
9
 Ideal Properties of
Detector
1. High sensitivity
2. Constant response for all
wavelengths
3. Fast response time
4. 0 response for no light
5. Signal directly proportional to
radiant power of light

10
 A. Photon Detectors

Commonly useful in ultraviolet, visible, and near

infrared instruments.

- Several types of photon detectors are

available:
1. Vacuum phototubes
2. Photomultiplier tubes
3. Photovoltaic cells
4. Silicon photodiodes
5. Photodiode array
6. Photoconductivity

11
B. Thermal Detectors

Used for infrared spectroscopy because photons in the IR region

lack energy to cause photoemission (produce electron) of
electrons.

Respond to heat.

IR radiation hits thermal transducer, will raise the temperature

slightly and this change in temperature, can be measured by
some change in the materials properties

Three types of thermal detectors:

1. Thermocouples - simplest
2. Bolometers/thermistors
3. Pyroelectric - extremely fast response
12
 IV. Sample Container
Usually called cells or cuvettes must have windows that are
transparent in the spectral region of interest.

There are few types of cuvettes:

- quartz or fused silica
- silicate glass
- crystalline sodium chloride

Quartz or fused silica

- required for UV and may be used in visible region

Silicate glass
Silicate glass cuvettes can be used in the visible only. Importan
Much cheaper than quartz t!

Crystalline sodium chloride

- Used in IR.
13
 Plastic (disposable) cuvettes can also be used in the
visible region and transmit light from 350-900 nm.

Most common path length in UV and Visible region is 1

cm.

Quality of spectroscopic data critically dependent on the

way cell are used and maintained.

Fingerprints, grease or other deposits on the walls

markedly alter the transmission characteristic of a cell.

Thorough cleaning before and after use and care must be

taken to avoid touching the windows after cleaning is
complete.
14
 SUMMARY
Types of source, sample holder and
detector
REGION
for various
SOURCE
EM region
SAMPLE DETECTOR
HOLDER
Ultraviolet Deuterium lamp Quartz/fused Phototube,
silica Photomultiplier
tube, diode
array
Visible Tungsten lamp Glass/quartz Phototube, PM
tube, diode
array

Infrared Nernst glower Salt crystal e.g. Thermocouples,

(rare earth crystal sodium bolometers
oxides or silicon chloride
carbide glowers) 15
2. ULTRAVIOLET-VISIBLE
SPECTROSCOPY

16
 In this lecture, you will
learn:
Absorption process in UV/VIS region
in terms of its electronic transitions
Molecular species that absorb UV/VIS
radiation
Important terminologies in UV/VIS
spectroscopy

17
 INSTRUMENTATION
Important components in a UV-Vis
spectrophotometer
5
1 2 3 4
Signal
Source Sample selector Detector processor
lamp holder
& readout
UV region:
-Deuterium lamp; Quartz/fused silica Prism/monochromator Phototube,
H2 discharge tube PM tube, diode
array

Visible region:
- Tungsten lamp Glass/quartz Prism/monochromator Phototube,
PM tube, diode
array

18
 Instrumentation
UV-Visible instrument
1. Single beam
2. Double beam

19
 SINGLE BEAM INSTRUMENT

Radiation from the filter or monochromator passes

through either the reference cell or the sample cell
before striking the photodetector.
 Single beam instrument

Disadvantages:

Two separate readings has to be made on the

light. This result in some error because the
fluctuations in the intensity of the light do occur
in the line voltage, the power source and in the
light bulb between measurements.

Changing of wavelength is accompanied by a

change in light intensity. Thus spectral scanning
is not possible.
21
 DOUBLE BEAM
INSTRUMENT

Radiation from the filter or monochromator is split into two

beams that simultaneously pass through the reference and
sample cells before striking two matched photodetectors.
 Double-beam instrument
March 2013

Advantages over single beam:

1. It is well suited for continuous

reading (scanning) of the absorption
spectra
2. It eliminate the need to alternate
blank and sample manually so that
frequency of error can be minimized.
23
 ORGANIC INORGANIC
COMPOUNDS SPECIES

MOLECULAR SPECIES THAT

ABSORB UV/VISIBLE
RADIATION

CHARGE
TRANSFER

24
 Definitions
Organic compounds
Chemical compound whose molecule contain carbon
E.g. C6H6, C3H4

Inorganic species
Chemical compound that does not contain carbon.
E.g. transition metal, lanthanide and actinide elements.
Cr, Co, Ni, etc

Charge transfer
A complex where one species is an electron donor and
the other is an electron acceptor.
E.g. iron (III) thiocyanate complex
25
PERIODIC TABLE OF
ELEMENTS

26
 ULTRAVIOLET-VISIBLE
SPECTROSCOPY
In UV/VIS spectroscopy, the
transitions which result in the
absorption of EM radiation in this
region are transitions between
electronic energy levels.

27
 Molecular absorption

In molecules, not only have

electronic level but also consists of
vibrational and rotational sub-levels.

This result in band spectra.

28
 Types of transitions

3 types of electronic transitions

- , and n electrons
- d and f electrons
- charge transfer electrons

29
 What is , and n electrons?
single covalent bonds ()

H + O + H H O H or H O H

lone pairs(n

O C O or O C O

double bonds ()

N N or N N

triple bond ()
30
 Sigma () electron

Electrons involved in single bonds such as

those between carbon and hydrogen in alkanes.

These bonds are called sigma () bonds.

The amount of energy required to excite

electrons in bond is more than UV photons of
wavelength. For this reason, alkanes and other
saturated compounds (compounds with only
single bonds) do not absorb UV radiation and
therefore frequently very useful as
transparent solvents for the study of other
molecules. For example, hexane, C6H14.
31
 Pi () electron

Electrons involved in double and triple

bonds (unsaturated).

These bonds involve a pi () bond.

For example: alkenes, alkynes and

aromatic compounds.

Electrons in bonds are excited

relatively easy; these compounds
commonly absorb in the UV or visible
region.
32
 Examples of organic molecules
containing bonds.
H
CH3 C C H

CH2CH3
propyne
H C H
C C
H H
C C
H C H H C C

C C H
H
ethylbenzene benzene H H

1,3-butadiene

33
 n electron

Electrons that are not involved in

bonding between atoms are called n
electrons.

Organic compounds containing nitrogen,

oxygen, sulfur or halogens frequently
contain electrons that are nonbonding.

Compounds that contain n electrons

absorb UV/VIS radiation.
34
 Examples of organic
..
molecules with
non-bonding electrons.
O:

: NH2 C H3C H
R
C C
..
: Br
.. H
Carbonyl compound
If R = H aldehyde 2-bromopropene
aminobenzene
If R = CnHn ketone

35
ABSORPTION BY ORGANIC COMPOUNDS

UV/Vis absorption by organic

compounds requires that the energy
absorbed corresponds to a jump from
occupied orbital to an unoccupied
orbital of greater energy.

Generally, the most probable transition

is from the highest occupied molecular
orbital (HOMO) to the lowest
unoccupied molecular orbital (LUMO).
36
 Electronic energy levels diagram

* Antibonding

Unoccupied levels
* Antibonding
*

n *

n *
*
Energy

n Nonbonding

Bonding
Occupied levels

Bonding

37
 Electronic transitions

* In alkanes

* In alkenes, carbonyl compounds, alkynes,

Increasing
energy n * In oxygen, nitrogen, sulfur and halogen
compounds

n * In carbonyl compounds

38
 Electronic transitions
* transitions

The energy required to induce a

* transition is large (see the arrow
in energy level diagram).

This type of absorption corresponds

to breaking of C-C, C-O, C-H, C-X,
.bonds
39
 n * transitions
- Saturated compounds containing atoms
with unshared electron pairs (non-bonding
electrons).
- Compounds containing O, S, N and
halogens can absorb via this type of
transition.
- Absorption are typically in the range, 150
- 250 nm region and are not very intense.
range: 100 3000 cm-1mol-1
- Absorption maxima tend to shift to shorter
in polar solvents.
e.g. H2O, CH3CH2OH
40
Some examples of absorption
due to
n * transitions
Compound max (nm) max
H2O 167 1480

CH3OH 184 150

CH3Cl 173 200

CH3I 258 365

(CH3)2O 184 2520

CH3NH2 215 600

41
 n * transitions

- Unsaturated compounds containing

atoms with unshared electron pairs
(nonbonding electrons)
- These result in some of the most intense
absorption in range, 200 700 nm
- Unsaturated functional group
- To provide the orbitals
range: 10 100 Lcm-1mol-1

42
 * transitions

- Compounds with unsaturated

functional groups to provide the
orbitals.
- These result in some of the most
intense absorption in range, 200
700 nm
range: 1000 10,000 Lcm-1mol-1

43
Examples n * and *

H O

H C C

H H

* at 180 nm
n * at 290 nm
44
 MOLECULAR SPECIES THAT ABSORB
UV/VISIBLE RADIATION

(A) Absorption by organic compounds

2 types of electrons are responsible:

i. Shared electrons that participate directly

in bond formation ( and bonding
electrons)

i. Unshared outer electrons (nonbonding or n

electrons)
45
 Absorption by organic compounds

The shared electrons in single bonds, C-C

or C-H ( electrons) are so firmly held.
Therefore, not easily excited to higher E
levels. Absorption ( *) occurs only in
the vacuum UV region ( 180 nm).

Electrons in double & triple bonds

(electrons) are more loosely held.
Therefore, more easily excited by
radiation. Absorptions ( *) for species
with unsaturated bonds occur in the
UV/VIS region ( 180 nm)
46
Absorption by organic compounds

CHROMOPHORES
Unsaturated organic functional
groups that absorb in the
UV/VIS region.

47
Typical organic functional groups
that serve as chromophores
Chromophores Chemical structure Type of transition

Acetylenic -CC- *

Amide -CONH2 *, n *

Carbonyl >C=O *, n *

Carboxylic acid -COOH *, n *

Ester -COOR *, n *

Nitro -NO2 *, n *

Olefin >C=C< *
48
49
 Absorption by organic
compounds
AUXOCHROME
Groups such as OH, -NH2 & halogens
that attached to the double bonded
atoms cause the normal
chromophoric absorption to occur at
longer (red shift).

50
 Effect of
Multichromophores
on Absorption
More chromophores in the same molecule cause
bathochromic effect ( shift to longer ) and
hyperchromic effect (increase in intensity).

51
 Absorption by organic
compounds
Factors that influenced the :

i) Solvent effects (shift to shorter :

blue shift)

ii) Structural details of the molecules

52
 Absorption spectra for
typical organic compounds

53
Important terminologies

Hypsochromic shift (blue shift)

- Absorption maximum shifted to
shorter

Bathochromic shift (red shift)

- Absorption maximum shifted to
longer

54
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

iii. Hyperchromic effect an increase in intensity

Hyperchromic
iv. Hypochromic effect a decrease in intensity

Hypsochromic Bathochromic
Hypochromic

200 nm 700 nm
55
 Terminology for Absorption
Shifts
Nature of Shift Descriptive Term
To Longer Bathochromic
Wavelength
To Shorter Hypsochromic
Wavelength
To Greater Hyperchromic
Absorbance
To Lower Hypochromic
Absorbance 56
 (B) Absorption by inorganic species

Involving d and f electrons absorption

(transition metal ions)

3d & 4d electrons
- 1st and 2nd transition metal series
e.g. Cr, Co, Ni & Cu
- Absorb broad bands of VIS radiation
(broad peaks)

57
Absorption spectra of some transition-metal ions
and rare earth ions

Most transition metal ions are colored (absorb in UV-VIS) due to d d

58
electronic transitions
 Absorption by inorganic
species
4f & 5f electrons
- Ions of lanthanide and actinide
elements
- Their spectra consists of sharp,
narrow, well-defined
characteristic absorption peaks.

59
 (C) Charge transfer absorption

Absorption involved transfer of electron

from the donor to an orbital that is
largely associated with the acceptor.

An electron occupying in a or orbital

(electron donor) in the ligand is
transferred to an unfilled orbital of the
metal (electron acceptor) and vice-versa.

e.g. red colour of the iron (III) thiocyanate

complex
60
 Many inorganic complexes of
electron donor (usually organic) &
electron acceptor (usually metal)
examples: Iron III thiocyanate
Iron II phenanthroline

61
Absorption spectra of aqueous charge transfer
complexes

62
 Quantitative Analysis

The fundamental law on which

absorption methods are based on
Beers Law (Beer-Lambert Law).

63
 Measuring Absorbance
You must always attempt to work at the
wavelength of maximum absorbance (max).

This is the point of maximum response, so

better sensitivity and lower detection limits.

You will also have reduced error in your

measurement.

64
65
 Quantitative Analysis

Calibration curve method

Standard addition method

66
 Calibration curve method

- A general method for determining

the concentration of a substance in
an unknown sample by comparing
the unknown to a set of standard
sample of known concentration.

67