INSTRUMENTAL METHODS UNIT-5

Electromagnetic Spectrum:
The electromagnetic Spectrum is the full range of electromagnetic radiation, organized by frequency
or wavelength.

The spectrum is divided into separate bands With different from Low to names for the
electromagnetic wave high frequency range is Radio waves to Gamma-rays.

Type of Radiation frequency range (13) WAVE LENGTH RANGE

Gamma-rays 1020-1024 <10-12m

x-rays 1017-1020 1nm-1pm

Visible 1015-1017 400nm-1nm

Neal IR 4x1014-7.5x1014 750nm-400nm

IR 1x1014-4x1014 2.5µm-750nm

Microwaves 1013-1014 25µm-2.5µm

Radio waves <3x1011 >1mm

Y (electric field)

Direction of propagation [OR]

Electric field

Propagation

Magnetic field
Absorption of radiation : (fundamental law)
Absorption of electromagnetic radiation is takes up a photons energy and so transform
electromagnetic energy into internal energy of absorber
Excited state

hv=E2-E1

Ground state

white light is mixture of all Sever colours (VIBGYOR), the absorption of any one of the colours from
white light results in the complementary colour being observed.

By measuring the decrease in intensity of radiation we can quantitatively and qualitatively

analyze a Substance This technique is called photometry.

fundamental Laws of Absorption :- There are two laws which govern the absorption of light by
molecules.

(a) Lamberts Law

(b) Beers Law

Lambert's Law:- when a beam of monochromatic [single colour light] is passed through a pure
homogenous absorbing medium. The rate of decrease in intensity of a radiation with the thickness
"Of absorbing medium is proportional to the intensity of incident Light `(Io)
𝒅𝑰
− ∝ 𝑰𝒐
𝒅𝒙

𝒅𝑰
− 𝒅𝒙 = 𝒌𝑰𝒐

𝒅𝑰
− 𝑰 = 𝒌. 𝒅𝒙
𝒐

Then ,
𝒅𝑰
𝑰𝒐
= −𝒌. 𝒅𝒙

Integrating equation between 𝑰 = 𝑰𝒐 to I & x=0 to x=x

𝑰 𝒅𝑰 𝒙=𝒙
∫𝑰𝒐 𝑰 = − ∫𝒙=𝟎 𝒌. 𝒅𝒙
𝒐

𝑰 𝒙
𝐥𝐨𝐠[𝑰] = −𝒌 ∫𝟎 𝒅𝒙
𝑰𝒐
𝒙
𝐥𝐨𝐠 𝑰 − 𝐥𝐨𝐠 𝑰𝒐 = −𝒌[𝒙]
𝟎
 𝑰
𝐥𝐨𝐠 = −𝒌𝒙
𝑰𝒐

Taking anti logs

𝑰
= 𝒆−𝒌𝒙
𝑰𝑶

𝑰 = 𝑰𝒐 . 𝒆−𝒌𝒙

𝑰𝒂𝒃𝒔 = 𝑰𝒐 − 𝑰

= 𝑰𝒐 − 𝑰𝒐 . 𝒆−𝒌𝒙

By changing natural log to base 10

From equation 4
𝑰
𝐥𝐨𝐠 𝑰 = −𝒌𝒙
𝒐

𝑰
𝟐. 𝟑𝟎𝟑 𝐥𝐨𝐠 𝟏𝟎 𝑰 = −𝒌𝒙
𝒐

𝑰 −𝒌
𝐥𝐨𝐠 𝟏𝟎 𝑰 = 𝟐.𝟑𝟎𝟑 𝒙
𝒐

𝑰 −𝒌
Where 𝐥𝐨𝐠 𝟏𝟎 𝑰 is called Absorbance and Represented by ‘A’ and 𝟐.𝟑𝟎𝟑 is called Absorbance Efficient
𝒐
and represented by ‘E’.

𝑨 = 𝑬𝒙 (At particular length 𝒙 = 𝒍)

𝑨 = 𝑬𝒍

This is lambert’s law. It says Absorbance of the incident is equal to multiple of absorbance co-
efficient and length of the particular.

BEER’S LAW [or ] BEER LAMBERT’S LAW :

When a beam of monochromatic light is passed through a solution of an absorbing medium,

The rate of decrease of intensity of radiation with thickness of absorbing solution is proportional to
the intensity of incident radiation as well as concentration of the solution.
𝒅𝑰
− 𝒅𝒙 ∝ 𝒄𝑰𝒐

𝒅𝑰
− 𝒅𝒙 = 𝑬𝒄𝑰𝒐

𝒅𝑰
𝑰𝒐
= −𝑬𝒄𝒅𝒙
Integrating equation both sides between limits 𝑰 = 𝑰𝒐 to I & x=0 to x=x
𝑰 𝒅𝑰 𝒙
∫𝑰𝒐 𝑰 = − ∫𝟎 𝑬𝒄𝒅𝒙
𝒐

𝑰 𝒙
𝐥𝐨𝐠[𝑰] = −𝑬𝒄 ∫𝟎 𝒅𝒙
𝑰𝒐
𝒙
𝐥𝐨𝐠 𝑰 − 𝐥𝐨𝐠 𝑰𝒐 = −𝑬𝒄[𝒙]
𝟎

𝐥𝐨𝐠 𝑰 − 𝐥𝐨𝐠 𝑰𝒐 = −𝑬𝒄𝒙

𝑰
𝐥𝐨𝐠 𝑰 = −𝑬𝒄𝒙
𝒐

By taking anti logs

𝑰
𝑰𝑶
= 𝒆−𝑬𝒄𝒙

𝑰 = 𝑰𝒐 . 𝒆−𝑬𝒄𝒙

Intensity of light absorbed is given by ,

𝑰𝒂𝒃𝒔 = 𝑰𝒐 − 𝑰

= 𝑰𝒐 − 𝑰𝒐 . 𝒆−𝑬𝒄𝒙

By changing natural logarithm to base 10

𝑰
𝟐. 𝟑𝟎𝟑 𝐥𝐨𝐠 𝟏𝟎 = −𝑬𝒄𝒙
𝑰𝒐

𝑰 𝟏
𝐥𝐨𝐠 𝟏𝟎 =− 𝑬𝒄𝒙
𝑰𝒐 𝟐.𝟑𝟎𝟑

𝑰
𝐥𝐨𝐠 𝟏𝟎 𝑰 = − 𝑬| 𝒄𝒙
𝒐

𝑰 |
= 𝟏𝟎−𝑬 𝒄𝒙
𝑰𝑶

|
𝑰 = 𝑰𝒐 . 𝟏𝟎−𝑬 𝒄𝒙
𝟏
𝑬| =
𝒙

𝟏
𝑰 = 𝟏𝟎 𝑰𝒐

𝑬| is the reciprocal of thickness of a solution layer molar concentration, which reduces the intensity
of light passing through it to 𝟏⁄𝟏𝟎 𝒕𝒉 of its original.
 LIMITATIONS OF BEER LAMBERT’S LAW :
1.This law obeyed for only monochromatic light

2.Applicable to only dilute solutions

3.The temperature of the system should remains constant (Because raise in temp shifts the
absorbtion bands towards lower wavelength ).

GRAPH:

ABSORBANCE
B .

CONCENTRATION

UV-VISIBLE SPECTROSCOPY:

Principle :

The branch of spectroscopy which deals with the interaction of UV-Visible radiation with
matter is called UV-Visible spectroscopy.

when a molecule absorbs Electro magnetic radiation in Uv- visible region (UV→150-400nm,
visible→400-700nm) it results electronic transitions. So it is also called as Electronic Spectroscopy

Electronic Transitions: The absorption of Electromagnetic radiation causes electrons to be excited,

which results in promotion from a bonding /non-bending orbitals to an anti-banding orbitals i.e -

There are four types of electronic Transitions 𝛔 − 𝛔 ∗ ,𝛑 − 𝛑∗

σ∗

σ ∗ [LUMO]
π∗

𝑛
↑↓
σ [HOMO]

σ
According to molecular orbital theory:
We have , Highest Occupied Molecular Orbital [HOMO]
Lowest Un-occupied Molecular Orbital[LUMO]

1. σ − σ ∗-transition:
In this transition electron excited from 'o' bonding molecular orbital to

𝛔 ∗ anti bonding molecular orbital.

It takes place in compounds having 𝛔 - bonds such as saturated hydrocarbons (Alkanes) & 𝑯𝟐 .

Ex: 𝑪𝑯𝟒 , 𝑪𝟐 𝑯𝟔 , 𝑪𝑯𝟑 − 𝑪𝑯𝟐 − 𝑪𝑯𝟑 , 𝑯𝟐 .

∗ ∗
σ [LUMO] σ [LUMO]

↓
ΔE(258kcal/mol
e) UV
↓↑ 111nm ↑

σ [HOMO] σ [HOMO]

2. π − π∗ transition: In this transition electron excited from π bonding molecules Orbital

to anti bonding molecular orbital.

It takes place in molecules containing π bonds. Such as unsaturated hydrocarbons.

Ex: 𝑪𝑯𝟐 = 𝑪𝑯𝟐 , 𝑪𝑯 ≡ 𝑪𝑯 , etc.

* (LUMO) AE (173 kcal/mode) (HOMO)
π[LUMO] π[LUMO]
↓
ΔE(173kcal/mol
e) UV
↓↑ 165nm ↑

π∗ [𝐻𝑂𝑀𝑂] π∗ [HOMO]

3. n- σ ∗ transition: In this transition electron excited from ‘n’ non-

bonding molecules orbital to σ ∗ anti bonding molecular orbital.

Ex: HX, NH3, 𝑪𝑯𝟑 𝑪𝑯𝟐 𝑶𝑯 , 𝑵𝑯𝟑 , 𝑯𝟐 𝑺,etc. wavelength=150nm

 4. η-π∗ Transition: In this transition electron exited From ‘η’ non-bonding molecular
∗
orbital to π antibonding molecular orbital At takes place in molecules Containing
multiple bond and tone pair of electrons.

ex- Aldehydes (RCHO), ketones (RLOR), RLOOH, RCOOR (esters) etc.

𝐶𝐻3
𝑯𝟑 𝑪 − 𝑪𝑶 − 𝑪𝑯 = 𝑪 ΔE = 91kcal/mole ; wave length=314nm
𝐶𝐻3

→ Increasing order of energies in electronic Transitions:-

1
η-π∗ < π − π∗< n- σ ∗ < σ − σ ∗ note : 𝐸 ∝
λ
 chromophores : chromophores is any group which responsible for imparting
colour to the compound

Chromophore Contain electron and undergo I-Tradit ext Carbonyl, nitriles ete.

→ Auxochromes : These are basically a colour enhancing group with ability to tend conjugation.

Ex : -OH, -OR - NH₂ , -Si, etc.

 Instrumentation : the UV-visible absorptions are measured by the instrument UV-

Spectrophotometer. The compounds when exposed to or light, which under go
different electronic transitions. The Absorbance can be given as -.
𝑰𝒐
𝑨 = 𝐥𝐨𝐠 𝟏𝟎 𝑰
where 𝑰𝒐 =Intensity of sample
𝑰 =Intensity of reference

 In UV Spectrophotometer a beam of light split into two equal halves.

 one half of the beam is diverted towards the Cell Containing a sort of compound bring
analyzed.

 Other half is directed towards the cell contains Solvent (Reference)

 the instrument is designed in such a way that intensities of two beams will be
compared at each Wavelengths.

 The Intensity of sample beam will be less than will be less than reference beam.

 out put graph is called Absorption Spectrum.

 mirror Data reading
Photo
Tungster lamp diode a
(source) reference
b
s
o
Data r
processing b
filter a
n
c Wave length
e
Photo
diode
Monochromater
Beam spliiter sample

Applications:

 UV-visible spectroscopy is the use of Beer – Lambert’s Law to determine concentration of

chromophores.

→ Molar extinction coefficient (E) is obtained.

→ the concentration of DNA sample is determined more Useful biochemists.

→Useful to predict existence of different groups like Conjugation, c=c, benzene rings etc

→ Useful to determine structure of several Vitamins, dissociation Constants of aids, Bases.

Comparents of Instruments
1. Source
2. Filter
3. Mono chromator
4. Beam splitter
5. Mirror
6. Reference call
7. Sample cell
8. Photo Diode
9. Processor (Amplifier)
10. Data Read on