UV-Visible spectroscopy

1. Seminar on
Ultra-Violet & Visible Spectroscopy
Presented by
Mounik Rout
M.Pharm (Pharmaceutical technology)
Guided by
Dr. Sasmita Kumari Acharjya
ROLAND INSTITUTE OF PHARMACEUTICAL SCIENCES

2. Contents
SPECTROSCOPY
WAVE NUMBER , FREQUENCY , ENERGY
EMR WAVELENGTH
UV-VISIBLE SPECTRUM
ELECTRONIC TRANSITION
CHROMOPHORE
AUXOCHROME
ABSORPTION
ABSORPTION LAWS
INSTRUMENTATION

3. Spectroscopy:-
• Study of absorption of Electromagnetic radiation by molecule, atom, or ion of a sample when it
excited from one energy state to another energy state .
• Electromagnetic radiation are used in spectroscopic method as a source of radiation .
• 1.Wavelength(λ) - The distance in between both the crests and troughs is wavelength .
• The unit used for wavelength are - Å , nm , μm .
Fig2. Detailed wave structure

4. • 2.Wavenumber(̄) – Number of waves passing through the space of 1cm is called
wavenumber .
• ̄= 1/λ
• The unit is Cmˉ¹
• 3.Frequency () - Number of waves passing through a point in duration of 1second is
called frequency of the EMR .
• Frequency = C/λ
• Value of C = 3 x10⁸ cm/sec
• Unit is Hz
Fig4. frequency

5. • 4.Energy(E) :- Electromagnetic radiation have some energy .
• When wavelength increases , energy of EMR decreases , & when wavelength
decreases energy of EMR increases .
• E = h (where h is planck’s constant=6.626 x 10ˉ²⁷ erg sec)
• E= h x C/λ (=C/λ)

6. Different EMR and their wavelength
Electromagnetic Radiation Wavelength
Gamma Ray <0.001nm
X-Rays 0.01-10nm
Ultra violet Rays 200-400nm
Visible Rays 400-800nm
Infra red Rays 0.8-200μm
Near IR 0.8-2.5μm
Mid IR 2.5-15μm
Far IR 15-200μm
Microwaves 0.01-1m
Radiowaves 1-10⁷m
Table.1

7. 1.30-
1.20-
1.10-
1.00-
0.90-
0.80-
0.70-
0.60-
0.50-
0.40-
0.30-
0.20-
0.10-
0.00-
200nm 300nm 400nm 500nm 600nm 700nm 800nm
Fig5. UV-Visible spectrum

8. Electronic Transition :-
• When molecule are excited from one energy state to higher energy state , electronic
transition takes place .
• Types of electrons involved in electronic transition ;
Type 1- Bonded electrons (e.g. 𝜎 , 𝜋 )
Type 2- Non bonded electrons (e.g. n)
Types of electronic transition ;
1. 𝝈 − 𝝈 ∗
2. n − 𝜎 ∗
3. 𝜋 − 𝜋 ∗
4. n − 𝝅 ∗ Decreasing order of energy requirement

9. • Electronic Transition:-
Lowest Unoccupied Molecular Orbital (LUMO)
σ ∗
π ∗
n
π
σ
Highest Occupied Molecular Orbital (HOMO)
Fig6 . Electronic Transition

10. • Electronic Transition :-
1. 𝝈 − 𝛔* - When electron in σ get excited to σ* then this transition is called 𝜎 − σ*transition .
• All the saturated hydrocarbons (e.g. methane , propane) comes under this .
• This transition require high energy than all 4 transition .
• so that we have to go for vacuum UV region which is below 200nm .
• Below 200nm oxygen starts getting absorb , so under vacuum UV region
it will be free from any else compound , and only the transition will occur .
2. n − 𝛔*- When electron in n get excited to 𝛔* then the transition is called n − 𝛔* transition .
• This transition occur when any heteroatom is present in the saturated compound .
• This transition requires energy less than 𝝈 − 𝛔* transition .
• Heteroatoms used as solvent in UV-Visible spectroscopy because they aren’t
getting absorb in UV-Visible range .

11. 3. 𝛑 − 𝛑* - When electron from 𝛑 molecular orbital get excited in 𝛑* orbital , the transition
called 𝛑 − 𝛑* transition .
• This transition requires less energy in comparison of n − 𝛔* transition .
• Any double/triple bonded hydrocarbons are present then 𝛑 − 𝛑* transition will occur .
4. n −𝛑* - When electron from n molecular orbital get excited in 𝛑* orbital , the transition
called n −𝛑* transition .
• It requires lowest energy as comparison of all 4 transition .

12. • Chromophore- These covalently bonded moieties with any compound which are responsible for
absorption of electromagnetic radiation in the UV-Visible region .
Example of chromophore : Aldehyde , Ethylene, Carbonyl .
1. Chromophore with 𝛑 − 𝛑* : ex - >C=C< , -C≡C-
2. Chromophore with n −𝛑* : ex - -N=N-
Whenever chromophore compound will added to any compound the absorption wavelength will
increase .
• Auxochrome – It is defined as any moieties which does not shows any absorption when separated
but when it combines with any chromophore it increases the wavelength towards longer wavelength
by
formation of a new chromophore .
ex: -OH , NH₂ ,OR , etc .

13. • Absorption :-
• It is a quantitative evaluation .
• Spectroscopy is based upon absorption theory.
Incident Radiation(Iₒ)
Transmitted Radiation (Iₜ)
• Various cases of absorption ;
• Case1 – Intensity of Iₜ = Iₒ
• Case2 – Intensity of Iₜ < Iₒ (shows that absorption occurred)
• Case3 – scattering , refraction , reflection occurs .
• To compensate scattering , Nephelo turbidimetre is used .

14. • Absorption laws :-
 Lambert’s Law :- when a beam of monochromatic radiation is passed through the absorbing medium
then the decrease in the intensity of radiation will directly proportional to the thickness of solution .
• Equation A = Logₗₒ (Iₒ/Iₗ) ∝ L
A ∝ L L= Thickness of solution / Thickness of cuvette
A = 𝜖 L 𝜖 = Molar absorptivity co-efficient (unit= l/mol.cm)
 Beer’s law :- When a beam of monochromatic radiation is passed through the solution then the
decrease in the intensity of radiation will be directly proportional to the concentration of the solution .
• Equation A = Logₗₒ (Iₒ/Iₗ) ∝ C
A ∝ C C = Concentration of the solution
A = ϵC

15. • Beer Lambert’s Law –
• When a beam of monochromatic radiation is passed through the absorbing medium ,
then the decrease in the intensity of radiation is directly proportional to the thickness
as well as the concentration of the solution .
Equation => A = Logₗₒ (Iₒ/Iₗ)∝ L.C
A ∝ L.C L= Thickness of cuvette
A = ϵ L.C ϵ = Molecular absorptivity coefficient

16. • .

17. Instrumentation of UV-Visible spectroscopy
• .
Radiation
Source Entrance slit
Lens
Collimating lens
Gratings
Exit slit
Mirror
Chopper
Reference
Sample
Detector 1
Detector 2
Amplifier
Recorder
Monochromator

18. • Instrumentation
• There are 2 type of spectrophotometer used in UV-Visible spectroscopy .
1. Single beam
2. Double beam
• Double beam spectrophotometer – The instruments used in double beam
spectrophotometer are ;
1. Radiation source
2. Monochromator : Entrance slit
Collimating lens
Gratings
Reflecting lens
Exit slit
3. Chopper 5. Cuvette
4. Mirror 6. Detector
7. Amplifier
8. Recorder

19. • Radiation source – 1. Hydrogen-Deuterium lamp (ranges from 200-375nm)
2. Tungsten lamp (ranges from 375 -800nm for uv-visible spectroscopy)
• Monochromator – used to convert polychromatic radiation beam to monochromatic
radiation .
• Collimating lens – This converts the non-parallel beams to parallel beam .
• Gratings – This is used in place of prism now-a-days because it contains 1200-1400 groves
per mm .
• Exit slit – through this the required wavelength of beam passes to Chopper .
• Chopper – It equally divide the radiating beam in 2 parts in to the mirror .
• Mirror – it reflects the radiation in to both Cuvette containing reference and sample .
• Cuvette – it is made up of Quartz and glass material .
1. Quartz is used because it have very smooth surface and it don’t absorb the UV
radiation .
2. Glass is used in visible spectroscopy & it has UV absorption capacity .

20. • Detector – Detector used to detect the wavelength coming through the cuvette & it is of 3 types :
1. Photomultiplier tube – It multiplies the photons rays of the radiation .
Amplifier connected from the cathode and anode and amplifies the radiation .
2. Photodiode Array – In this detector there is presence of diodes , which converts
radiation into electronic signal .
• It is mainly used in HPLC , and its range is up to 200-800nm .
Photon
Electron
Dynode
fig. Photomultiplier tube

21. • 3. Charged coupled diode – Silicon surface is used to catch photon from the radiation of
UV-Visible source & converts radiation in to digital signal .
• Recorder – It records the amplified wavelength and shows the UV-Visible spectrum .
Single beam spectrophotometer – This is slightly different as comparison to double beam ,
It directly radiates the incident radiation coming from exit slit of monochromator to the
sample cuvette and then detector detects the radiation to the amplifier . Then the amplified
data recorded on the recorder as spectrum .

22. • Reference :
Text book of Pharmaceutical Analysis by
Dr. S. Ravi Sankar ,

23. THANK YOU