1. Analytical methods
Prepared By
Dr. Biswajit Saha

2. Spectroscopy
Spectroscopy is the branch of science which deals with the determination of the structure of a compound through a the interactions of various types of electromagnetic radiation with the matter.
What is electromagnetic radiation ?
Example: Light
Light interacting with matter
Exactly how the radiation interacts with matter is directly dependent on the energy of the radiation.
Energy , E= hv
Wave length, frequency etc.

3. Electromagnetic Spectrum Electromagnetic Waves

4. Visible region Violet: 400 - 420 nm Indigo: 420 - 440 nm
Blue:   nm 
Green:   nm 
Yellow:   nm 
Orange:   nm 
Red:   nm

5. Energy level diagram Different colors observed when the white
light was dispersed through the prism
Energy level diagram E2
vibrational levels v2 v1
rotational levels r2 v0 r1 E1
Pure electronic transition
Eelec >> Evib >> Erot v2
DE = hn v1 r2 v0 E0 r1
electronic levels 5

6. Absorption & Emission
Electron exist in a certain number of possible states corresponding to definite amounts of energy.
Electron can absorb energy and change to a higher energy level called the excited state.
The amount of energy absorbed in this transition is exactly equal to the energy difference between the states.

7. E2 E1 E0 ENERGY Energy Level Diagram for an Atom of Sodium
590 nm
330 nm E0
Energy Level Diagram for an Atom of Sodium
Ground State

8. Atomic energy level

9. Different Spectroscopies
UV-vis – electronic states of valence e/d-orbital transitions for solvated transition metals
Fluorescence – emission of UV/vis by certain molecules
IR – Vibrational transitions of molecules
X-Ray Spectroscopy – Electronic transitions of core electrons
NMR – Nuclear spin transitions

10. UV-Visible Spectroscopy
Ultraviolet-visible spectroscopy involves the absorption of ultraviolet/visible light by a molecule causing the promotion of an electron from a ground electronic state to an excited electronic state.
Ultraviolet/Visible light: wavelengths (l) between 190 and 800 nm
UV-visible spectrum
The two main properties of an absorbance peak are:
Absorption wavelength lmax
Absorption intensity Amax

11. UV-Visible Spectroscopy
When a beam of monochromatic radiation passes through a homogeneous
medium, A part of the incident light is reflected, a part of it is absorbed
within the medium and the remainder is transmitted. Let the intensity of
Incident light (I0), absorbed light (Ia ), transmitted light (It) and
Reflected light (Ir). Then
I0=Ia+It+Ir
106 photons
0.7x106 photons 
500 nm

13. Beer-Lambert Law and UV-Visible Spectroscopy
Lambert Law: the rate of the intensity of light decrease with thickness of the medium is proportional to the Intensity of the incident light.
-dI/dx α I
Beer-Lambert Law: the rate of the intensity of light decrease with thickness of the medium is proportional to the Intensity of the incident light as well as concentration of the solution.
-dI/dx α cI
Mathematical derivation x

14. Beer-Lambert Law log(I0/I) = exc A = exc I A = log = e xc = - log T I
I0 = Intensity of incident light
I = intensity of transmitted light
= molar absoptivity coefficient in cm2 mol-1
c = concentration in mol L-1
x = pathlength of absorbing solution in cm-1
A = absorbance = log(Io/I)
T=transmittance (I/I0)
log(I0/I) = exc
A = exc I A =
log = e xc = -
log T I

15. Transmittance, Absorbance, and Concentration

16. Numerical problem
The percentage transmittance of an aqueous solution of unknown compounds is 20 % at 25 oC and 300 nm for 4x10-5 M solution in a 2 cm cell.
The absorbance (A)
The molar extinction or absorption coefficient
Ans , 8.7x103 M-1 cm-1
For a solution of camphor in hexane in a 10 cm cell, the absorbance (A) was found to be 2.52 at 295 nm with molar absorptivity (ε) 14. What is the concentration of the camphor?

17. Limitation of Beer-Lambert Law

18. Five types of transitions are possible: *, *, n *,  * n *
Molecular energy level
Five types of transitions are possible:
*,
*,
n *,
 *
n *

19. Effect of conjugation on UV spectra
The addition of conjugation in
naphthalene, anthracene and
tetracene causes bathochromic
shifts of these absorption bands,

20. Lycopene from Tomatoes
Visible region

21. UV-visible spectroscopy definitions
Chromophore: Any group of atoms that absorbs light whether or not a color is thereby produced.
Auxochrome : A group which extends the conjugation of a chromophore by sharing of nonbonding electrons.
Bathochromic shift: The shift of absorption to a longer wavelength. Because the red color in the visible spectrum has a longer wavelength than most other colors, this effect is also commonly called a red shift.
Hypsochromic shift : The shift of absorption to a shorter wavelength. (blue shift)