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
200nm

Visible
400nm

Wavelength (nm)

IR
800nm

Microwave

Electromagnetic Radiation
V = Wave Number (cm-1)
l = Wave Length
C = Velocity of Radiation (constant) = 3 x 1010 cm/sec.
u = Frequency of Radiation (cycles/sec)

The energy of photon:

h (Planck's constant) = 6.62 x 10- (Ergsec)
27

E = hu= h

C
l

u =

C
l

C = ul

Spectral Properties, Application and Interactions of

Electromagnetic Radiation
Wave
Number V

Energy

Wavelength

Kcal/mol

eV

cm-1

cm

9.4 x 107

4.9 x 106

3.3 x 1010

3 x 10-11

9.4 x 103

9.4 x 101

4.9 x 102

4.9 x 100

3.3 x 106

3.3 x 104

3 x 10-7

3 x 10-5

Frequency

Type
Radiation

Type
spectroscopy

Type
Quantum Transition

Gamma
ray

Gamma ray
emission

Nuclear

Hz
1021

1017

X-ray

1015

Ultra
violet

X-ray
absorption,
emission

Electronic
(inner shell)

UV absorption

Electronic
(outer shell)

Visible
IR absorption

9.4 x 10-1

4.9 x 10-2

3.3 x 102

3 x 10-3

1013

Infrared

9.4 x 10-3

4.9 x 10-4

3.3 x 100

3 x 10-1

1011

Microwave

Microwave
absorption

Radio

Nuclear
magnetic
resonance

9.4 x 10-7

4.9 x 10-8

3.3 x 10-4

3 x 103

107

Molecular
vibration

Molecular
rotation

Magnetically
induced spin
states

Dispersion of Polymagnetic Light with a Prism

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

monochromatic
Ray

Polychromatic
Ray

PRISM

Red
Orange
Yellow
Green
Blue
Violet

Ultraviolet

Polychromatic Ray

Monochromatic Ray

SLIT

Beer Lambert Law

Light
I0

Glass cell filled with

concentration of solution (C)

As the cell thickness increases, the transmitted intensity

of light of I decreases.

T- Transmittance
T=

I
I0

I0 - Original light intensity

I- Transmitted light intensity
I
I0

% Transmittance = 100 x

Absorbance (A) = Log

1
T

= Log

I0

= 2 - Log%T

I
Log

I0
I

is proportional to C (concentration of solution) and is

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

E =

Absorbance x Liter
Moles x cm

UNITS
A = ECL
A = No unit (numerical number only)
Liter
E =
Cm x Mole

L = Cm

C = Moles/Liter

A = ECL = (

Liter
Cm x Mole

)x

Mole
Liter

x Cm

Steps in Developing a Spectrometric Analytical Method

2. Obtain a monochromatic
wavelength for the maximum
absorption wavelength.
3. Calculate the concentration of
your sample using Beer Lambert
Equation: A = ECL

Absorbance

1. Run the sample for spectrum

2.0

0.0
200

250

300

350

400

Wavelength (nm)

450

Spectrometer Reading

A
C

Slope of Standard Curve =

1.0
x

0.5

4
2
3
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

0.8

0.4

3
1
2
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.

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 , , 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.

n *

n *

Antibonding

Energy

Antibonding

Nonbonding
Bonding
Bonding

Electronic Molecular Energy Levels

The higher energy transitions ( *) occur a

shorter wavelength and the low energy transitions
(*, n *) occur at longer wavelength.

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)

Visible Spectrometer Application

Niacin
Pyridoxine
Vitamin B12
Metal Determination (Fe)
Fat-quality Determination (TBA)
Enzyme Activity (glucose oxidase)

Vibrasi IR

Vibrasi IR

Vibrasi IR

Tipe-tipe Vibrasi

Rocking

Scissorring

Asym streching Sym streching

Twisting

Wagging