UV Visible spectroscopy.pptx

Ultraviolet–visible spectroscopy, Its working principle and Applications.

1. Ultraviolet–visible spectroscopy Presented by: Mr. Sanjoy Chungkrang Lecturer , Department of Pharmaceutical science Northeast Frontier Technical University, Alo, Arunachal Pradesh

2. UV VISIBLE SPECTROSCOPY

3. UV VISIBLE SPECTROSCOPY DEFINATION :- UV- Visible spectroscopy is a quantative analysis technique which is used to measure the attenuation of a beam of light (UV) after it passed through a sample or reflectant from a sample surface .

4. Light source Sample Incident light Transmitted Light (100 nm) (60nm) Absorption = Incident Light – Transmitted Light

5. PRINCIPLE The UV- Visible spectroscopy is work based on Absorption spectroscopy. The Principle is explained by two laws – BEER’S LAW LAMBERT’S LAW

6. BEER’S LAW The decrease in the intensity of light is directly proportional to the concentration. OR The absorption of uv radiation is directly proportional to the concentration

7. Light source Sample Incident light Transmitted Light A α C (i)

8. LAMBERT’S LAW According to Lambert’s law the decrease in the intensity of light is directly proportional to path length travelled by the light. OR The absorption is directly proportional to the path length.

9. Light source Sample Incident light Transmitted Light A α b (ii) (Path length)

10. BEER’S LAMBERT’S LAW From Equation (i) and (ii) , we find that- A α c (i) A α b (ii) Therefore, A α C b A= € C b Here, A = Absorption of a given wave length. € = Molar absorbity Constant. C = Concentration of the solution. b = Path length, Distance light passed.

11. INSTRUMENTATION & IT’S WORKING Monochromator

12. Instrumentation and working of the UV spectrometers can be studied simultaneously. Most of the modern UV spectrometers consist of the following parts- LIGHT SOURCE : Tungsten filament lamps and Hydrogen-Deuterium lamps are most widely used and suitable light source as they cover the whole UV region. Tungsten filament lamps are rich in red radiations; more specifically they emit the radiations of 375 nm, while the intensity of Hydrogen-Deuterium lamps falls below 375 nm . Deuterium lamp

13. MONOCHROMATOR - Monochromators generally composed of prisms and slits. The most of the spectrophotometers are double beam spectrophotometers. The radiation emitted from the primary source is dispersed with the help of rotating prisms. The various wavelengths of the light source which are separated by the prism are then selected by the slits such the rotation of the prism results in a series of continuously increasing wavelength to pass through the slits for recording purpose. The beam selected by the slit is monochromatic and further divided into two beams with the help of another prism PRISM

14. SAMPLE & REFERENCE CELL - One of the two divided beams is passed through the sample solution and second beam is passé through the reference solution. Both sample and reference solution are contained in the cells. These cells are made of either silica or quartz. Glass can't be used for the cells as it also absorbs light in the UV region. I II III Fig :- Cuvette / Sample

15. DETECTOR Generally two photocells serve the purpose of detector in UV spectroscopy. One of the photocells receives the beam from sample cell and second detector receives the beam from the reference. The intensity of the radiation from the reference cell is stronger than the beam of sample cell. This results in the generation of pulsating or alternating currents in the photocells.

16. RECORDING DEVICES Most of the time amplifier is coupled to a pen recorder which is connected to the computer. Computer stores all the data generated and produces the spectrum of the desired compound.

17. APPLICATION OF UV- VISIBLE SEPCTROSCOPY- It is used in a quantative analysis . It is used to detect extended conjugation Conjugated molecule are stable due to resonance and have low energy, so it gives higher value of λ max (absorption maximum) then Non- conjugated. Example:- Penta-1,3-diene (conjugated) CH2 = CH – CH = CH - CH3 Penta-1,4-diene (Non- conjugated) CH2 = CH – CH – CH = CH2

18. It is used to detect both Aromatic and Non- Aromatic Compound. Aromatic compound are resonance stabilizer, so they give higher value of λ max then Non- aromatic compound. Example :- Benzene Cyclohexane (High λ max) (low λ max)

19. It is useful to detect Geometrical Isomers: - (a) Cis – Isomers (b) Trans - Isomers Cis isomers are less stable due to high energy and repulsion than trans – isomers, so trans – isomers have higher value of λ max than Cis – isomers.

20. Example :- (High λ max) (Low λ max)

21. And also the UV- Visible spectroscopy is used in various activity such as – Detection of Impurities ( Organic Solvent). Drug Identification. Bacterial Culturing. Determination of Molecular weight by using Beer’s Law.