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MOLECULAR ABSORPTION

SPECTROSCOPY :
THEORY,
INSTRUMENTATION &
APPLICATION
CHAPTER 2
COMPONENTS OF
INSTRUMENTS FOR
OPTICAL SPECTROSCOPY
General Design of Optical
Instruments
Absorption
Emission
Five Basic Optical Instrument Components
1) Source A stable source of radiant energy at the desired
wavelength (or range).

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 EM spectrum used for measurement
(monochromators, prisms & filters).

4) Detector/Photoelectric Transducer Converts the radiant
energy into a useable signal (usually electrical).

5) Signal Processor & Readout Amplifies or attenuates the
transduced signal and sends it to a readout device as a
meter, digital readout, chart recorder, computer, etc.
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.
- Tungsten lamp is the most common visible source.
- Glowing inert solids are common sources for IR
instruments.
B. Line Sources Emit a limited number lines or
bands of radiation 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 amplification by stimulated
emission of radiation
II. Wavelength Selectors
Wavelength selectors output a limited,
narrow, continuous group of wavelengths
called a band.

Two types of wavelength selectors:
1) Filters
2) Monochromators
A. Filters
- Two types of filters:
1) Interference Filters
2) Absorption Filters

B. Monochromators
- Wavelength selector that can continuously
scan a broad range of wavelengths

- Used in most scanning spectrometers
including UV, visible, and IR instruments.
III. Radiation Transducer (Detectors)
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.

Two general types of radiation transducers:
a. Photon detectors
b. Thermal detectors
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. Diode array transducers
6. Photoconductivity transducers

B. Thermal Detectors

- Used for infrared spectroscopy
because photons in the IR region lack
energy to cause photoemission of
electrons.

- Three types of thermal detectors:
1. Thermocouples
2. Bolometers
3. Pyroelectric transducers

IV. Sample Container
Sample containers, 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
- Cheaper compared to quartz. Used in UV.

Crystalline sodium chloride
- Used in IR.



Spectrometer
- is an instrument that provides information
about the intensity of radiation as a function
of wavelength or frequency.

Spectrophotometer
- is a spectrometer equipped with one or
more exit slits and photoelectric transducers
that pemits the determination of the ratio of
the radiant power of two beams as a function
of wavelength as in absorption spectroscopy.
SUMMARY
Types of source, sample holder and detector
for various EM region
REGION SOURCE SAMPLE
HOLDER
DETECTOR
Ultraviolet Deuterium lamp Quartz/fused silica Phototube, PM
tube, diode array
Visible Tungsten lamp Glass/quartz Phototube, PM
tube, diode array

Infrared Nernst glower
(rare earth oxides
or silicon carbide
glowers)
Salt crystal e.g.
crystal sodium
chloride
Thermocouples,
bolometers
ULTRAVIOLET-VISIBLE
SPECTROSCOPY
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
In this lecture, you will learn:
INSTRUMENTATION
Important components in a UV-Vis spectrophotometer
Source
lamp
Sample
holder
selector Detector
Signal
processor
& readout
1 2 3 4
5
UV region:
-Deuterium lamp;
H
2
discharge tube
Visible region:
- Tungsten lamp

Glass/quartz

Prism/monochromator

Phototube,
PM tube, diode
array

Quartz/fused silica

Prism/monochromator

Phototube,
PM tube, diode
array
Instrumentation
UV-Visible instrument
1. Single beam
2. Double beam
Single beam instrument
Single beam instrument

- One radiation source
- Filter/monochromator ( selector)
- Cells
- Detector
- Readout device
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 btw measurements.

Changing of wavelength is accompanied by a
change in light intensity. Thus spectral
scanning is not possible.

Double beam instrument
Double-beam instrument with beams separated in space
Double-beam instrument

Advantages:
1. Compensate for all but most short-term
fluctuations in the radiant output of the
source as well as for drift in the
transducer and amplifier.
2. Compensate for wide variations in
source intensity with .
3. Continuous recording of transmittance
or absorbance spectra.
MOLECULAR SPECIES THAT
ABSORB UV/VISIBLE RADIATION
INORGANIC
SPECIES
ORGANIC
COMPOUNDS
CHARGE
TRANSFER
Definitions:
Organic compounds
Chemical compound whose molecule contain carbon
E.g. C
6
H
6
, C
3
H
4

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
PERIODIC TABLE OF
ELEMENTS
In UV/VIS spectroscopy, the transitions
which result in the absorption of EM
radiation in this region are transitions
between electronic energy levels.


ULTRAVIOLET-VISIBLE
SPECTROSCOPY
In molecules, not only have electronic
level but also consists of vibrational and
rotational sub-levels.

This result in band spectra.
Molecular absorption
Types of transitions
3 types of electronic transitions
- , and n electrons
- d and f electrons
- charge transfer electrons
H H
O
+ + O H H O H H or
single covalent bonds ()
O C O or O C O
double bonds ()
N N N N
triple bond ()
or
What is , and n electrons?
lone pairs(n)
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, C
6
H
14
.



Electrons involved in double and triple
bonds (unsaturated).
These bonds involve a pi () bond.
For exampel: alkenes, alkynes,conjugated
olefins and aromatic compounds.
Electrons in bonds are excited relatively
easily; these compounds commonly
absorb in the UV or visible region.
Pi () electron
Examples of organic molecules containing
bonds.
CH
2
CH
3
C
C
C
C
C
C
H
H
H
H
H
H
C H
C
CH
3
ethylbenzene
benzene
propyne
C
H
C
C
H
H C
H H
H
1,3-butadiene
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 re nonbonding.
Compounds that contain n electrons
absorb UV/VIS radiation.
n electron
Examples of organic molecules with non-
bonding electrons.
NH
2
C
O
R
C
C
H
H Br
H
3
C
:

..
:
..
..
:
aminobenzene
Carbonyl compound
2-bromopropene If R = H aldehyde


If R = C
n
H
n
ketone
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).
ABSORPTION BY ORGANIC COMPOUNDS
Electronic energy levels diagram
Antibonding
Antibonding
Nonbonding
Bonding
Bonding
Unoccupied levels
Occupied levels


n
*
*
E
n
e
r
g
y

*

n

*

n

*

Electronic transitions
*
*
n *
n *
In alkanes
In alkenes, carbonyl compounds, alkynes, azo
compounds
In oxygen, nitrogen, sulfur and halogen
compounds
In carbonyl compounds
Increasing
energy
* transitions

The energy required to induce a *
transition is large (see the arrow in energy
level diagram).

Never observed in the ordinarily accessible
ultraviolet region.

This type of absorption corresponds to
breaking of C-C, C-O, C-H, C-X, .bonds
Electronic 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
-1
mol
-1
- Absorption maxima tend to shift to shorter in polar
solvents.
e.g. H
2
O, CH
3
CH
2
OH
n * transitions
Some examples of absorption due to
n * transitions
Compound
max
(nm)
max

H
2
O 167 1480
CH
3
OH 184 150
CH
3
Cl 173 200
CH
3
I 258 365
(CH
3
)
2
O 184 2520
CH
3
NH
2
215 600
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
-1
mol
-1

* 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
-1
mol
-1

Examples n * and *
C
C
O
H
H
H
H
* at 180 nm
n * at 290 nm
(A) Absorption by organic compounds

2 types of electrons are responsible:
i. Shared electrons that participate directly
in bond formation ( and bonding
electrons)
ii. Unshared outer electrons (nonbonding
or n electrons)
MOLECULAR SPECIES THAT ABSORB
UV/VISIBLE RADIATION
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)
Absorption by organic compounds
CHROMOPHORES
Unsaturated organic functional
groups that absorb in the UV/VIS
region.
Absorption by organic compounds
Typical organic functional groups
that serve as chromophores
Chromophores Chemical structure Type of transition
Acetylenic -CC- *
Amide -CONH
2
*, n *

Carbonyl >C=O *, n *

Carboxylic acid -COOH *, n *

Ester -COOR *, n *

Nitro -NO
2
*, n *

Olefin >C=C< *

Groups such as OH, -NH
2
& halogens
that attached to the double bonded atoms
cause the normal chromophoric absorption
to occur at longer (red shift).

Absorption by organic compounds
AUXOCHROME
Effect of Multichromophores
on Absorption
More chromophores in the same molecule
cause bathochromic effect ( shift to longer )
and hyperchromic effect (increase in
intensity).
In conjugated chromophores, * electrons are
delocalized over larger number of atoms.
This cause a decrease in the energy of *
transitions and an increase in due to an
increase in probability for transition.
Factors that influenced the :

i) Solvent effects (shift to shorter : blue
shift)

ii) Structural details of the molecules
Absorption by organic compounds
Absorption spectra for typical organic
compounds
Hypsochromic shift (blue shift)
- Absorption maximum shifted to shorter

Bathochromic shift (red shift)
- Absorption maximum shifted to longer
Important terminologies
Nature of Shift Descriptive Term
To Longer Wavelength Bathochromic
To Shorter Wavelength Hypsochromic
To Greater Absorbance Hyperchromic
To Lower Absorbance Hypochromic
Terminology for Absorption Shifts
(B) Absorption by inorganic species

Involving d and f electrons absorption

3d & 4d electrons
- 1
st
and 2
nd
transition metal series
e.g. Cr, Co, Ni & Cu
- Absorb broad bands of VIS radiation
- Absorption involved transitions between
filled and unfilled d-orbitals with energies that
depend on the ligands, such as Cl
-
, H
2
O, NH
3

or CN
-
which are bonded to the metal ions.
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
electronic transitions
4f & 5f electrons
- Ions of lanthanide and actinide elements
- Their spectra consists of narrow, well-
defined characteristic absorption peaks.
Absorption by inorganic species

(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

Absorption spectra of aqueous charge transfer
complexes
The fundamental law on which absorption
methods are based on Beers Law (Beer-
Lambert Law).
Quantitative Analysis
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.
Measuring Absorbance

Calibration curve method
Standard addition method
Quantitative Analysis
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.
Standard Calibration Curve
A
b
s
o
r
b
a
n
c
e

How to measure the concentration of unknown?
Practically, you have measure the absorbance of your
unknown. Once you know the absorbance value, you can just
read the corresponding concentration from the graph.
How to produce standard calibration curve
Prepare a series of
standard solution with
known concentration.

Measure the absorbance of
the standard solutions.

Plot the graph Abs vs
concentration of std.

Find the best straight line.
Stock solution
100 ppm
Calibration standard
A
b
s
o
r
b
a
n
c
e


The slope of the line, m:

m = y
2
y
1
x
2
x
1


The intercept, b:

b = y mx

Thus, the equation for the least-square line is:

y = mx + b



Concentration, x y = mx + b
5
10
15
20
25
From the least-square line equation, you can calculate
the new y values by substituting the x value.

Then plot the graph.
Standard addition method

- used to overcome matrix effect
- involves adding one or more increments
of a standard solution to sample aliquots
of the same size.
- Each solution is diluted to a fixed volume
before measuring its absorbance.
A
b
s
o
r
b
a
n
c
e


Standard Addition Plot
How to produce standard
addition curve?
1. Add same quantity of unknown sample to a series of flasks.

2. Add varying amounts of standard (made in solvent) to each
flasks, e.g. 0, 5, 10, 15 mL).

3. Fill each flask to line, mix and measure.
Standard Addition
Methods
Single-point standard
addition method
Multiple standard
addition method
Standard addition
- if Beers Law is obeyed,

A = bV
std
C
std
+ bV
x
C
x

V
t
V
t


= kV
std
C
std
+ kV
x
C
x


k is a constant equal to b
V
t

Standard Addition
- Plot a graph: A vs V
std

A = mV
std
+ b

where the slope m and intercept b are:

m = kC
std
; b = kV
x
C
x
C
x
can be obtained from the ratio of these
two quantities: m and b

b = kV
x
C
x

m kC
std


C
x
= bC
std

mV
x

10 ml aliquots of raw-water sample were
pipetted into 50.0 ml volumetric flasks. Then,
0.00, 5.00, 10.00, 15.00 and 20.00 ml
respectively of a standard solution containing
10 ppm of Fe
3+
were added to the flasks,
followed by an excess of aqueous potassium
thiocyanate in order to produce the red iron-
thiocyanate complex. All the resultant
solutions were diluted to volume and the
absorbance of each solution was measured
at the same.
Example:
Vol. of std added
(ml)
Absorbance
(A)
0 0.215
5.00 0.424
10.00 0.625
15.00 0.836
20.00 1.040
The results obtained:
Calculate the concentration of Fe
3+
(in ppm)
in the raw-water sample
0
0.2
0.4
0.6
0.8
1
1.2
-10 -5 0 5 10 15 20 25
A
b
s
o
r
b
a
n
c
e

Vol. of std
Absorbance vs Vol. of std added
Slope, m = 0.0382
b = 0.24
(V
std
)
0
= -6.31 ml
Note: From the graph, extrapolated value represents the volume of
reagent corresponding to zero instrument response.
The unknown concentration of the analyte
in the solution is then calculated:

C
sample
= -(V
std
)
0
C
std

V
sample


C
x
= bC
std

mV
x

The chromium in an aqueous sample was determined by pipetting
10.0 ml of the unknown into each of 50.0 mL volumetric flasks.
Various volumes of a standard containing 12.2 ppm Cr were added
to the flasks, following which the solutions were diluted to the mark.
SELF-EXERCISE
Volume of
unknown (mL)
Volume of
standard (mL)
Absorbance
10.0 0.0 0.201
10.0 10.0 0.292
10.0 20.0 0.378
10.0 30.0 0.467
10.0 40.0 0.554
i) Plot a suitable graph to determine the concentration of Cr in the
aqueous sample.
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.
400 500 600 800 700
, nm
Violet 400-420
Indigo 420-440
Blue 440-490
Green 490-570
Yellow 570-585
Orange 585-620
Red 620-780
Visible Spectroscopy
When white (continuum of )
light passes through, or is
reflected by a surface, those s
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.

Visible Spectroscopy
Organic compounds that are colored are typically those with
extensively conjugated systems (typically more than five).
Consider b-carotene.
b-carotene,
max
= 455 nm

max
is at 455 nm in the far blue
region of the spectrum . This is
absorbed.
The remaining light has the
complementary color of orange.
Visible Spectroscopy

max
for lycopene is at 474 nm in the near blue region of
the spectrum this is absorbed, the compliment is now red.

max
for indigo is at 602 nm in the orange region of the
spectrum. This is absorbed, the compliment is now indigo!
lycopene,
max
= 474 nm
N
H
H
N
O
O
indigo
Visible Spectroscopy