U1 C1 UV Spectroscopy.pptx

UV-Visible spectroscopy, a versatile analytical technique used to study the interaction of molecules with light within the ultraviolet and visible regions of the electromagnetic spectrum. It is a fundamental tool in chemistry, biochemistry, and related fields for analyzing the electronic structure of molecules, determining their concentrations, and studying their behavior.
1. Principle of UV-Visible Spectroscopy:
UV-Vis spectroscopy is based on the principle that molecules absorb specific wavelengths of light due to electronic transitions.
When molecules absorb light in the UV or visible range, they move from a ground state to an excited state.
2. Instrumentation:
UV-Vis spectrophotometer is the key instrument used for this technique.
Components include a light source, sample holder, monochromator, and a detector.
The sample is placed in a cuvette, and the spectrophotometer measures the absorbance of light passing through the sample.
3. Beer-Lambert Law:
The Beer-Lambert law relates the concentration of a solution, the path length (distance that light travels through the solution), and the absorbance of light by the solution.
A = ε * c * l, where A is absorbance, ε is the molar absorptivity (a constant for a specific compound and wavelength), c is the concentration, and l is the path length.
4. Absorbance Spectra:
UV-Vis spectroscopy generates absorbance spectra, which are plots of absorbance versus wavelength.
Peaks in the spectra indicate the wavelengths of light that are absorbed by the sample, providing information about the electronic structure of the molecules.
5. Applications:
Quantitative Analysis: UV-Vis is widely used for quantitative analysis of compounds by measuring the absorbance of a sample and comparing it to a standard curve.
Identification of Compounds: The unique absorbance spectra can be used to identify compounds.
Kinetics: UV-Vis can monitor reaction kinetics by following the change in absorbance over time.
Pharmaceutical Analysis: It is crucial in quality control for pharmaceuticals.
Environmental Analysis: UV-Vis is used in environmental monitoring, such as water quality analysis.
6. Advantages:
It's a rapid and simple technique.
It can be highly sensitive for many compounds.
It is non-destructive to the sample.
7. Limitations:
It does not provide structural information about the molecules.
It may not be suitable for analyzing complex mixtures.

1. UNIT –I CHAPTER -1 UV- VISIBLE SPECTROSCOPY By- Prof. Ankita Raikwar Department of Analysis SOPS/JNU

2. CH-1 UV Spectrophotometer Introduction Principle- Beer Lambert Law Absorbance ∝ Conc. & Path length Instrumentation Application

3. Table of contents Spectroscopy UV VIS Spectroscopy UV Visible Spectrum Electronic Transitions Types of electronic transitions Chromophores Auxochromes Spectral shifts Solvents used in UV VIS Spectroscopy Factors affecting the position of UV bands Principle Beer and Lambert’s law Instrumentation of UV visible Spectrophotometer Applications

4. SPECTROSCOPY branch of science that deals with the study of interaction of electromagnetic radiation with matter principle is based on the measurement of spectrum of a sample containing atoms / molecules spectrum is a graph of intensity of absorbed or emitted radiation by sample verses frequency (ν) or wavelength (λ) study of interaction of EMR with matter used for analysis of wide range of samples Spectrometer- instrument designed to measure the spectrum of a compound. Spectroscopy is a necessary tool for structure determination.

5. Spectrum

6. UV-VIS SPECTROSCOPY absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region analytical method that can measure the analyte quantity depending on the amount of light received by the analyte ultraviolet-visible spectral field refers to absorption spectroscopy or reflectance spectroscopy. In the visible and neighbouring (near-UV and near-infrared (NIR) ranges, this means that it uses light.

7. Far/ vaccum uv region (10-200nm) near or quartz uv region (200-400nm) visible region (400-780nm)

8. based on the absorption of ultraviolet light or visible light by chemical compounds when electrons are promoted to higher energy levels, which results in the production of distinct spectra. deals with the measurement of energy absorbed when electrons are promoted to higher energy levels. involves electronic transitions, it is often called electronic spectroscopy. plot of wavelength of light absorbed versus the absorption intensity (absorbance or transmittance) and is conveniently recorded by plotting molar absorptivity (ε) against wavelength (nm).

9. Electronic Transitions electrons in organic molecules are involved in bonding as strong bonds, or weak bonds or present in the non-bonding form (lone pair) Hence, variety of energy absorptions for electronic transitions within a molecule is thus possible depending upon the nature of bonding.

10. Types of electronic   transitions A) Transitions between bonding and antibonding orbitals : 2 Types- ( i ) σ to σ* Transition(120-200nm) : The excitation between bonding sigma and antibonding sigma orbitals (σ to σ* transition) requires large energies. For example, methane, propane, cyclohexane etc., display σ to σ* transitions λͅmax for each of these compounds is below 140 nm. (ii) π to π* Transition (170-190 nm): The transition or promotion of an electron from a π bonding orbital to a π* antibonding orbital. These type of transitions occur in compounds containing one or more covalently unsaturated groups like C=C, C≡C, C=O, N=N, aromatic rings, NO2, etc. In unconjugated alkenes,; ethylene shows λͅmax at around 171 nm.

11. (ii) n to σ* Transition : The excitation of an electron in an unshared pair (nonbonding electrons) on nitrogen, oxygen, sulphur or halogens to an antibonding σ orbital is called n to σ* transition. transition is of moderate intensity located around 180nm for alcohols, near 190 nm for ethers or halogen derivatives and in the region of 220 nm for amines. (iii) σ to π* Transition and π to σ* Transition : These electronic transition are forbidden transitions and are theoretically possible.

12. B) Transitions between non-bonding atomic orbitals and antibonding orbitals (200nm<) : 2 types ( i ) n to π* Transition : Transitions between non-bonding atomic orbitals holding unshared pair of electrons and antibonding pi- orbitals are called n to π* transitions. Non-bonding electrons are held more loosely than σ bonding electrons and consequently undergo transitions at comparatively longer wavelengths. For ex-compounds containing double bonds involving hetero atoms bearing unshared pair(s) of electrons (e.g., C=S, N=O etc.).

13. Chromophores system responsible for imparting colour to a compound. functional group that absorbs electromagnetic radiation, a 'color' is thereby produced. functional group containing multiple bonds capable of absorbing radiations above 200nm due to n to π* and π to π* examples are: C=O, C=C, C=N, N=N, C ≡ C, C ≡ N, C = S etc.

14. Auxochrome functional groups attached to a chromophore which modifies the ability of the chromophore to absorb light, altering the wavelength or intensity of absorption. auxiliary group which interacts with the chromophore causing a bathochromic shift. enhances the colour imparting properties of a chromophore without being itself a chromophore . examples are: methyl, hydroxyl, alkoxy , halogens, and amino and substituted amino groups (OR,NH2, NHR and NR2).

15. Spectral Shifts Bathochromic shift (Red Shift): shift to lower energy or longer wavelength. caused by change of medium (π to π* transitions undergo bathochromic shift with an increase in the polarity of the solvent) or when auxochrome is attached to C=C double bond for example, ethene absorbs at λͅmax = 175 nm while 1-butene absorbs at λͅmax = 190 nm. bathochromic shift is progressive as the number of alkyl groups increases.

16. Hypsochromic shift (Blue Shift): shift to higher energy or shorter wavelength. caused by change of medium (n to π* transitions undergo hypsochromic shift with an increase in the polarity of the solvent) for example, acetone absorbs at 280 nm in hexane and at 265 nm in water. shift results from hydrogen bonding which lowers the energy of n orbital of oxygen atom. This can also be produced when an auxochrome is attached to double bonds e.g. C=O) where n electrons are available.

17. Hypochromic effect: It is the effect leading to decreased absorption intensity, for example, benzoate ion has less absorption intensity than benzoic acid.

18. Hyperchromic effect: effect leading to increased absorption intensity. If auxochrome is introduced to a compound, the intensity of absorption increases. For example, phenolate ion has more absorption intensity than phenol.

19. Solvents used in UV VIS Spectroscopy 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

20. Factors affecting the  position of UV bands 1. Effect of solvent: choice of solvent can shift peaks to shorter or longer wavelengths. depends on the nature of the interaction of the particular solvent with the environment of the chromophore in the molecule Water and alcohols can form hydrogen bonds which results the shifting of the bands of polar substances. Since polarities of the ground and excited state of a chromophore are different, hence a change in the solvent polarity will stabilize the ground and excited states to different extent causing change in the energy gap between these electronic states. Highly pure , non-polar solvents such as saturated hydrocarbons “do not “ interact with solute molecules either in the ground or excited state

21. ( i ) π to π* Transitions: excited states are more polar than the ground state. If a polar solvent is used the dipole–dipole interaction reduces the energy of the excited state more than the ground state. Thus a polar solvent decreases the energy of π to π* transition and hence the absorption in a polar solvent such as ethanol will be at a longer wavelength (red shift) than in a non-polar solvent such as hexane. (ii) n to π* transitions: polar solvents form hydrogen bonds with the ground state of polar molecules more readily than with their excited states. energy of ground state is decreased which further causing increase in energy difference between the ground and excited energy levels Therefore, absorption maxima resulting from n to π* transitions are shifted to shorter wavelengths (blue shift ) with increasing solvent polarity.

22. 2. Effect of Sample pH: The absorption spectra of certain aromatic compounds such as phenols and anilines change on changing the pH of the solution. Phenols and substituted phenols are acidic and display sudden changes in their absorptions maxima upon the addition of a base. After removal of the phenolic proton, we get phenoxide ion. In the phenoxide ion lone pairs on the oxygen is delocalized over the π-system of the aromatic ring and increases the conjugation. Extended conjugation leads to a decrease in the energy difference between the HOMO and LUMO orbitals , which results in red or bathochromic shift , along with an increase in the intensity of the absorption.

23. aromatic amine gets protonated in an acidic medium which disturb the conjugation between the lone pair on nitrogen atom and the aromatic π-system. As a result, blue shift or hypsochromic shift (to shorter wavelength) is observed along with a decrease in intensity.

24. Principle based on the fundamental law of absorption called Beer-Lambert’s law. absorption of radiation by an absorbing medium (dilute solution)

25. Beer’s law states that the intensity of a beam monochromatic light decreases exponentially with the concentration of the absorbing molecules. absorbance ∝ concentration

26. Lambert’s law states that the intensity of a beam monochromatic light decreases exponentially as the light travels through a thickness of homogeneous medium absorbance ∝ path length But since dimension of cuvettee is fixed hence Lambert Law is quite nullified.

27. Instrumentation of UV  Visible spectrophotometer A) Sources of radiation: Stable provide sufficient intensity of radiation supply continuous radiation over the entire wavelength. Hydrogen/deuterium discharge lamp: consists of hydrogen gas under relatively high pressure through which there is an electrical discharge. -hydrogen molecules are excited electrically and emit UV radiation. -which causes the hydrogen to emit a broad band( continous ) rather than a simple hydrogen line spectrum. -The lamps are stable, robust, and widely used. -If deuterium (D,) is used instead of hydrogen, the emission intensity is increased.

28. ii . Tungsten filament lamp: similar in its functioning to an electric light bulb. -heated electrically to white heat. -It has 2 shortcomings. The intensity of radiation at short wavelengths (350 nm < ) is small. -to maintain a constant intensity, the electrical current to the lamp must be carefully controlled. -lamps are generally stable, robust, and easy to use. iii. Mercury arc: mercury vapour is under high pressure, and the excitation of mercury atoms is done by electric discharge. -a standard source for much ultraviolet work, is generally not suitable for continuous spectral studies because of the presence of sharp lines or bands.

29. iv. Xenon discharge lamp: xenon gas is stored under pressure in the range of 10-30 atmospheres. -xenon lamp possesses two tungsten electrodes separated by about 8 mm. - when an intense arc is formed between two tungsten electrodes by applying a low voltage, the ultraviolet light is produced. -intensity of ultraviolet radiation produced by xenon discharge lamp is much greater than that of hydrogen lamp.

30. B) Monochromator (wavelength selectors): used to disperse the radiation according to the wavelength. essential elements of a monochromator are an entrance slit, a dispersing element and an exit slit. entrance slit sharply defines the incoming beam of heterochromatic radiation. dispersing element disperses the heterochromatic radiation into its component wavelengths exit slit allows the nominal wavelength together with a band of wavelengths on either side of it. position of the dispersing element is always adjusted by rotating it to vary the nominal wavelength passing through the exit slit. The dispersing element may be a prism or grating. The prisms are generally made of glass, quartz fused silica. Glass has the highest resolving power but it is not transparent to radiations having the wavelength between 200 and 300 nm, because glass absorbs strongly in this region. Quartz and fused silica prisms which are transparent throughout the entire UV range are widely used in UV spectrophotometers.

31. C)Sample containers/sample cells/ Cuvettes : The cells that are to contain samples for analysis should fulfill these main conditions: uniform in construction thickness must be constant surfaces facing the incident light must be optically flat. material of construction should be inert to solvents. must transmit light of the wavelength used. made of quartz or fused silica. cells of pathlength 1cm are used.

32. D) Detectors i ) Photomultiplier tube: photomultiplier tube is a combination of a photodiode and an electron multiplying amplifier. consists of an evacuated tube which contains one photo-cathode and 9-16 electrodes known as dynodes. When radiation falls on a metal surface of the photocathode, it emits electrons. The electrons are attracted towards the first dynode which is kept at a positive voltage. When the electrons strike the first dynode, more electrons are emitted by the surface of dynode; these emitted electrons are then attracted by a second dynode where similar type of electron emission takes place. The process is repeated over all the dynodes present in the photomultiplier tube until a shower of electrons reaches the collector. number of electrons reaching the collector is a measure of the intensity of light falling on the detector. The dynodes are operated at an optimum voltage that gives a steady signal.

33. ii) Photo voltaic cell (Barrier Layer Cell): consists of a semiconductor, such as selenium, which is deposited on a strong metal base, such as iron. Then a very thin layer of silver or gold is sputtered over the surface of the semiconductor to act as a second collector electrode. radiation falling on the surface produces electrons at the selenium silver interface. barrier exists between the selenium and iron which prevents the electrons from flowing into iron. electrons are therefore accumulated on the silver surface. accumulation of electrons on the silver surface produces an electrical voltage difference between the silver surface and the base of cell. If external circuit has a low resistance, a photocurrent will flow which is directly proportional to the intensity of incident radiation beam.

34. iii) Photo tube (Photocell): It consists of a high-sensitive cathode in the form of a half-cylinder of metal which is contained in an evacuated tube. anode is also present in the tube which is fixed more or less along the axis of the tube. inside surface of the photocell is coated with a light sensitive layer. light is incident upon a photocell, the surface coating emits electrons. These are attracted and collected by an anode. current, which is created between the cathode and anode, is regarded as a measure of radiation falling on the detector. iv) Silicon Photodiode: A silicon photodiode is a solid-state device which converts incident light into an electric current. It consists of a shallow diffused p-n junction, normally a p-on-n configuration. (p-n junction is an interface or a boundary between two semiconductor material types, namely the p-type and the n- type, inside a semiconductor. The p-side or the positive side of the semiconductor has an excess of holes and the n-side or the negative side has an excess of electrons.)

35. Applications 1) Qualitative Analysis: presence or absence of certain functional groups 2) Detection of Impurities: for example, benzene as impurity in ethanol 3) Quantitative analysis: directly or by making calibration curve 4) Pharmaceutical analysis: Overlap of absorbance peaks in uv spectra can be used to find out the pharmaceutical compounds using mathematical derivatives. Chlortetracycline (antibiotic) and benzocaine (anesthetic) are identified simultaneously in veterinary powder formulation using first mathematical derivative. 5) Determination of strength of H-bonding: It is based on shift observed in polar solvents, for example, acetone absorbs at 279 (121kcal/mole) & 264.5 (126kcal/mole) nm resp. in hexane and water

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