UV Visible spectroscopy, introduction, principles, applications

V Visible spectroscopy, introduction, principles, applications

1. UV Visible spectroscopy NIVEDITHA G 7 th sem BPharm RR college of pharmacy

2. Ultraviolet and visible Spectroscopy is primarily used to measure the multiple bond or aromatic conjugation within the molecule. The absorption of the radiation is due to the subtraction of energy from the radiation beam when electrons in the orbital of lower energy are excited into orbital of higher energy. UV/Visible Spectroscopy requires electromagnetic radiation of high energy. Electromagnetic radiation:- EMR are form of energy consists of discrete pockets of energy moving with definite velocity. h- planks const. planks eqn E= hʋ E-energy absorbed v- freqency of light Introduction

3. Ultra-Violet radiation :- Ultra-violet means Beyond violet The wavelength range of uv radiation starts at blue end of visible light(4000Å) & ends at 2000A˚. REGION WAVELENGH(nm) Near Ultraviolet region (NUV) 400-200nm UVA or Long wave 400-320nm UVB or Medium wave 320-280nm UVC or Short wave Below -280nm Far Ultraviolet (FUV) 210-10nm Extreme Ultraviolet (EUV) 31-1nm

4. Principles involved in UV and Visible spectroscopy Any molecules has either ɳ , π and σ or combination of these electrons and these electrons absorb characteristic radiation and undergo transition from ground state to excited state. By characteristic absorption peaks depends on nature of electron present & based on this molecular stature can be evaluated.

5. Laws governing absorption of radiation:- Beer’s law Lambert’s law These two laws are applicable under the following condition: I o I a I t I r

6. Thus; I = Ia + It + Ir I = intensity of incident light Ia = intensity of absorbed light It = intensity of transmitted light Ir = intensity of reflected light But, reflected radiation – very small Therefore, I = Ia + It

7. Beer’s law: - states that ‘the intensity of a beam of monochromatic light decreases exponentially with increase in the concentration of absorbing species arithmetically’. Lambert’s law:- states that ‘when a beam of light is allowed to pass through a transparent medium, rate of decrease of intensity (I) with the thickness (t) of medium is directly proportional to the intensity of light’.

8. Mathematical derivation of Beer’s law:- - dI / dc α I - dI / dc = kI ……..(1) Where I denotes the intensity of incident light of λ wavelength, c denotes the concentration of medium and k denotes the proportionality factor. On integrating (1) and substituting I = I₀ when c =0 we get In I₀ / It = k c ̄ k1c It = Io e …….(2) I₀ = intensity of incident light It = intensity of transmitted light. K = absorption co-efficient for the given wavelength, C = concentration

9. ̄ k2b It = Io e ………. (3) b = path length Combining (2) & (3) ̄ € cb It = Io e ………. (4) log Io / It = € cb A = € cb ( log Io / It = A ) Where, C = concentration b = thickness of medium As per Lambert's law:-

10. Limitations of Beer’s law:- Low concentrations Not for suspensions Not valid – coagulation it cause scattering of radiation , leads to increase or decrease in absorbance value Monochromatic light should be used Measured absorbance lies b/n – 0.7 to 0.2 units(20-60 % T for better results.)

11. Deviations from beer’s law:- a = positive deviation b = obey’s of Beer’s law c = negative deviation

12. Deviation of Beer-Lambert Law Physical (true) deviation - high concentration - other absorbing species (increase A) Chemical deviation - dissociation of molecule in different pH - association, complexing , hydrolysis etc Instrumental deviation - polychromatic radiation - stray radiation, fluctuations

13. Physical deviation Concentration in 10 -6 M to 10 -7 M ( holds good) And others absorbing species – solvents and other molecules I I t

14. Chemical deviation Dissociation of molecules in different pH If the absorbing species under go chemical changes such as association, complex formation or hydrolysis. Presence of impurities. Reaction of absorbing species with each others or with solvents results in the formation of new species and these having different absorptivity . Ex: Dichromate ion shows deviation upon due to colour change (i.e., orange to yellow)

15. Solvent Effect:- The suitable solvent is one that does not itself get absorbed in the region under investigation. A solvent should be transparent in a particular region. A dilute solution of sample is always prepared for analysis. Most commonly used solvents are as follows . Solvent λ of absorption Water 191 nm Ether 215 nm Methanol 203 nm Ethanol 204 nm Chloroform 237 nm Carbon tetrachloride 265 nm Benzene 280 nm  Tetrahydrofuran 220 nm

16. Instrumental deviation It obeys when manochromatic radiation is used. Polychromatic radiation leads to negative deviation. Fluctuation in intensity of radiation. And changes in the detector sensitivity cause deviation. Improper slit width

17. Chromophore Chromophore ( chroma - colour, phoros - bearer) Covalently unsaturated group responsible for absorption ultra-violet or visible radiation. The colour usually appears in an organic compound if it contains unsaturated groups. A compound containing a Chromphore is called a Chromogen. Functional group which is responsible for imparting colour to the compound called chromophore . E.g.:- Nitro compounds are yellow in colour due to the presence of –NO 2 group as Chromophore.

18. Auxochromes:- An auxochrome can be defined as any group which does not itself act as chromophore but whose presence brings about a shift of the absorption band towards the red end of the spectrum ( longer wavelength). Some common auxochromic groups are -OH , -OR, -NH 2 , -NHR,-NR 2 , -SH, -SO 3 , -COOH

19. Types of shifts:- 1 . Bathochromic shift 2. Hypsochromic shift 3. Hyperchromic effect 4. Hypochromic effect Isobestic point:- It is a specific wavelength at which the molar absorption coefficient €max or two (or more) chemical substances in equilibrium.

20. Wavelength( λ) Absorbance ( Emax ) Red shift Blue shift Hypochromic shift Hyperchromic shift

21. Bathochromic shift :- ( red shift) It is an effect by virtue of which the absorption maximum is shifted towards longer wavelength due to presence of auxochrome (OH, NH 2 , OR) or by the change of solvent. E.g.:- 1,3-Butadiene which contains 2 conjugated double double bonds shows λmax at 217nm, Ethene which contains unconjugated double bond shows λmax at 170n m

22. Hypsochromic shift :- (blue shift) It is an effect by virtue of which the absorption maximum is shifted towards shorter wavelength due to change in polarity of solvent. E.g.:-In acidic solution exhibit blue shift Aniline λmax = 280nm Anilinium cation λmax = 203nm

23. Hyperchromic shift:- An effect due to which the intensity of absorption maximum increases i.e., ɛ max increases. The introduction of an auxochrome usually increase intensity of absorption. E.g.:- Pyridine λmax = 257nm λmax = 262nm 2-methyl pyridine

24. Hypochromic shift:- It is an effect due to which the intensity of absorption maximum decreases. The introduction of group which distorts the geometry of the molecule cause hypochromic effect . E.g.:- Biphenyl absorbs at 250 nm ( ɛ max 19000) whereas 2-methyl biphenyl absorbs at 237 nm ( ɛ max 10250).

25. Energy σ * ╥* n ╥ σ σ - σ * n- σ * ╥_╥* n - ╥* Bonding Bonding Nonbonding Antibonding Antibonding Electronic transition

26. 1. σ -- σ *Transition:- A transition of electron from a bonding sigma orbital to the higher energy anti-bonding sigma orbital. Sigma bonds are , in general very strong. Therefore is a high energy process and these transition require very short wavelength. E.g. Saturated Hydrocarbon. Energy σ * ╥* n ╥ σ σ - σ * Bonding Bonding Nonbonding Antibonding Antibonding

27. 2. n-- σ *Transition: This transition Occurs in saturated Compound containing one heteroatom with Unshared pair of involves (n-electrons). Corresponding absorption bands appear at longer wavelengths in near UV region, transition occur at 180 – 220 nm. e.g. Saturated Halides, Alcohol, Ethers, Aldehyde , and Amine. Energy σ * ╥* n ╥ σ n- σ * Bonding Bonding Nonbonding Antibonding Antibonding

28. 3. ╥ -- ╥* Transition:- This transition occurs in Unsaturated centers of the molecule. The excitation of ╥ electron requires smaller energy of longer wavelength. E.g .compound containing double or triple bond. i.e., alkenes, alkynes, nitrites Energy σ * ╥* n ╥ σ ╥_╥* Bonding Bonding Nonbonding Antibonding Antibonding

29. 4. n -- ╥* Transition:- These type of transition shown by unsaturated molecules which contains hetero atom such as oxygen, Nitrogen, and sulphur . This type transition requires lowest energy due to longer wavelength. Eg ., Aldehyde ketones The transition occurs at 270-350 nm. Energy σ * ╥* n ╥ σ n - ╥* Bonding Bonding Nonbonding Antibonding Antibonding

30. Applications: Detection of Impurities. Structure elucidation of organic compounds. Quantitative analysis. Qualitative analysis.

31. Reference:- Chatwal G.R., Instrumental Methods of Chemical Analysis, 5 th Edition, Himalaya Publishing House. Sharma B.K., Instrumental Methods for Chemical Analysis, 22th Edition, Krishna Prakashan Media Pvt. Ltd. A.V Kasture ., Pharmacutical analysis ( vol -II), Nirali Publication. Sharma Y.R., Elementry Organic Spectroscopy, 1 st Edition, S.Chand & Company Ltd.