SPECTROSCOPY

Introduction of Spectrometric Analyses

The study how the chemical compound

interacts with different wavelenghts in a given
region of electromagnetic radiation is called
spectroscopy or spectrochemical analysis.

The collection of measurements signals

(absorbance) of the compound as a function of
electromagnetic radiation is called a spectrum.
 Energy Absorption

The mechanism of absorption energy is different in

the Ultraviolet, Infrared, and Nuclear magnetic
resonance regions. However, the fundamental
process is the absorption of certain amount of energy.

The energy required for the transition from a state of lower

energy to a state of higher energy is directly
related to the frequency of electromagnetic radiation
that causes the transition.
Spectral Distribution of Radiant Energy

Wave Number (cycles/cm)

X-Ray UV Visible IR Microwave

200nm 400nm 800nm

Wavelength (nm)
 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- (Erg× sec)
27

C C
E = hυ
=h υ= C=
λ λ
υ λ
 Spectral Properties, Application and Interactions of
Electromagnetic Radiation

Wave Wavelength Frequency

Energy Number V λ υ
Type Type Type
Radiation spectroscopy Quantum Transition
Kcal/mol eV cm-1 cm Hz
9.4 x 107 4.9 x 106 3.3 x 1010 3 x 10-11 1021
Gamma Gamma ray
ray Nuclear
emission

9.4 x 103 4.9 x 102 3.3 x 106 3 x 10-7 1017 X-ray Electronic
X-ray
absorption, (inner shell)
emission
Ultra
9.4 x 101
4.9 x 10 0
3.3 x 10 4
3 x 10 -5
1015
violet UV absorption Electronic
Visible (outer shell)

9.4 x 10-1 4.9 x 10-2 3.3 x 102 3 x 10-3 1013 Infrared IR absorption Molecular
vibration Molecular
rotation
9.4 x 10-3 4.9 x 10-4 3.3 x 100 3 x 10-1 1011 Micro- Microwave
wave absorption
Magnetically
Nuclear induced spin
Radio magnetic
9.4 x 10-7 4.9 x 10-8 3.3 x 10-4 3 x 103 107 states
resonance
Spectrum of Radiation
 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

 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

• •
• •

σ
and σ
* orbitals π
and π
* orbitals
 Spectrophotometer

An instrument which can measure the absorbance of a

sample at any wavelength.

L ig h t L en s S lit M o n o ch ro m ato r S lits

S am p le D etecto r Q u an titativ e A n aly sis

 Fluorometer
Instrument to measures the intensity of fluorescent light emitted by a sample
exposed to UV light under specific conditions.

Emit fluorescent light Antibonding

as energy decreases σ'
π' Antibonding
n->σ' n->
π'
n Nonbonding
Ground state π −>π'
π Bonding
Energy σ −>σ
'
σ Bonding
Electron's molecular energy levels
UV Light Source Detector

Monochromator Monochromator
90°C

Sample
Food Compound

H3C S CH2 CH2 CH3

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

• •
• •

σ
and σ
* orbitals π
and π
* orbitals
 Beer – Lambert Law

Light
I0 I

Glass cell filled with

concentration of solution (C)

As the cell thickness increases, the transmitted intensity

of light of I decreases.
 R- Transmittance
I
R= I0 - Original light intensity
I0
I- Transmitted light intensity
 
I
% Transmittance = 100 x
I0

1
Absorbance (A) = Log
T
I0
= Log = 2 - Log%T
I
I
Log is proportional to C (concentration of solution) and is
I0
also proportional to L (length of light path
through the solution).
A ∝ CL = ECL by definition and it is called the
Beer - Lambert Law.
A = ECL

A = ECL
E = Molar Extinction Coefficient ---- Extinction
Coefficient of a solution containing 1g molecule of
solute per 1 liter of solution
 Absorbance x Liter
E =
Moles x cm

UNITS
A = ECL
A = No unit (numerical number only)

Liter
E =
Cm x Mole
L = Cm
C = Moles/Liter

Liter Mole
A = ECL = ( )x x Cm
Cm x Mole Liter
 Steps in Developing a Spectrometric Analytical Method

1. Run the sample for spectrum

2. Obtain a monochromatic
2.0
wavelength for the maximum

Absorbance
absorption wavelength.
3. Calculate the concentration of 0.0

your sample using Beer Lambert 200 250 300 350 400 450

Wavelength (nm)
Equation: A = ECL
Spectrometer Reading
 ∆
A
Slope of Standard Curve =
∆
C

A at 280 nm
1.0
x

0.5
x

1 2 3 4 5
Concentration (mg/ml)

There is some A vs. C where graph is linear.

NEVER extrapolate beyond point known where
becomes non-linear.
 Spectrometric Analysis Using Standard Curve

1.2

A at 540 nm
0.8

0.4

1 2 3 4
Concentration (g/l) glucose

Avoid very high or low absorbencies when drawing a standard

curve. The best results are obtained with 0.1 < A < 1. Plot the
Absorbance vs. Concentration to get a straight line
 Sample Cells

UV Spectrophotometer
Quartz (crystalline silica)

Visible Spectrophotometer
Glass
 Light Sources

UV Spectrophotometer
1. Hydrogen Gas Lamp
2. Mercury Lamp
Visible Spectrophotometer
1. Tungsten Lamp
Chemical Structure & UV Absorption

Chromophoric Group ---- The groupings of the

molecules which contain the electronic system which
is giving rise to absorption in the ultra-violet region.
 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
UV Spectrometer Application

Protein
Amino Acids (aromatic)
Pantothenic Acid
Glucose Determination
Enzyme Activity (Hexokinase)
Flurometric Application

Thiamin (365 nm, 435 nm)

Riboflavin
Vitamin A
Vitamin C
Visible Spectrometer Application

Niacin
Pyridoxine
Vitamin B12
Metal Determination (Fe)
Fat-quality Determination (TBA)
Enzyme Activity (glucose oxidase)
 Practice Examples
1. Calculate the Molar Extinction Coefficient E at 351 nm for
aquocobalamin in 0.1 M phosphate buffer. pH = 7.0 from the
following data which were obtained in 1 Cm cell.
Solution C x 10 M
5
Io I
A 2.23 100 27
B 1.90 100 32

2. The molar extinction coefficient (E) of compound

riboflavin is 3 x 103 Liter/Cm x Mole. If the absorbance reading
(A) at 350 nm is 0.9 using a cell of 1 Cm, what is the
concentration of compound riboflavin in sample?
 3. The concentration of compound Y was 2 x 10 moles/liter and
-4

the absorption of the solution at 300 nm using 1 Cm quartz cell

was 0.4. What is the molar extinction coefficient of compound
Y?

4. Calculate the molar extinction coefficient E at 351 nm for

aquocobalamin in 0.1 M phosphate buffer. pH =7.0 from the
following data which were obtained in 1 Cm cell.
Solution C x 10 M 5
I0 I
A 2.0 100 30
 Spectroscopy Homework
1. A substance absorbs at 600 nm and 4000 nm. What type of energy
transition most likely accounts for each of these absorption
processes?

2. Complete the following table.

[X](M) Absorbance Transmittance(%) E(L/mole-cm) L(cm)
30 2000 1.00
0.5 2500 1.00
2.5 x 10-3 0.2 1.00
4.0 x 10-5 50 5000
2.0 x 10-4 150
[X](M) = Concentration in Mole/L
3. The molar absorptivity of a pigment (molecular weight 300)
is 30,000 at 550 nm. What is the absorptivity in L/g-cm.

4. The iron complex of o-phenanthroline (Molecular weight

236) has molar absorptivity of 10,000 at 525 nm. If the
absorbance of 0.01 is the lowest detectable signal, what
concentration in part per million can be detected in a 1-cm
cell?