UV visible spectroscopy

UV visible spectroscopy

1. Prepared by- Mr.Akshay V. Patil M.pharm 1 st sem Dr, Rajendra gode college of pharmacy Malkapur . Guide by- Mr. P .V. Burukle sir Associate professor Dept. Pharamaceutical Analysis. Dr,Rajendra Gode college of pharmacy Malkapur . UV- visible Spectroscopy.

2. Introduction. Ultraviolet and visible (UV-Vis) absorption spectroscopy is the measurement of the attenuation of a beam of light after it passes through a sample or after reflection from a sample surface. Absorption measurements can be at a single wavelength or over an extended spectral range. The UV radiation region extends from 10 nm to 400 nm.

3. Near UV Region: 200 nm to 400 nm Far UV Region: below 200 nm Far UV spectroscopy is studied under vacuum condition. the visible radiation region extends from 400 nm to 800 nm. Ultraviolet absorption spectra arise from transition of electron with in a molecule from a lower level to a higher level. A molecule absorb ultraviolet radiation of frequency (𝜗), the electron in that molecule undergo transition from

4. lower to higher energy level. The energy can be calculated by the equation, E=h𝜗 erg Absorption spectrum When a sample is exposed to light energy that matches the energy difference between a possible electronic transition within the molecule, a fraction of the light energy would be absorbed by the molecule and the electrons would be promoted to the higher energy state orbital. A spectrometer records the degree of absorption by a sample at different wavelengths and the resulting plot of absorbance (A) .

5. versus wavelength (λ) is known as a spectrum. The significant features: λmax (wavelength at which there is a maximum absorption) єmax (The intensity of maximum absorption)

6. Every time a molecule has a bond, the atoms in a bond have their atomic orbitals merged to form molecular orbitals which can be occupied by electrons of different energy levels. Ground state molecular orbitals can be excited to anti-bonding molecular orbitals . These electrons when imparted with energy in the form of light radiation get excited from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO) and the resulting species is known as the excited state or anti-bonding state.

7. TYPES OF TRANSITIONS: In U.V spectroscopy molecule undergo electronic transition involving σ, π and n electrons.  Four types of electronic transition are possible. σ ⇾ σ* transition n ⇾ σ* transition n ⇾ π* transition π ⇾ π* transition

8. σ ⇾ σ* transition An electron in a bonding σ orbital of a molecule is excited to the corresponding anti-bonding orbital by the absorption of radiation. To induce a σ ⇾ σ* transition it required LARGE ENERGY. Ex: Methane Methane contain only single C-H bonds. it undergo only σ ⇾ σ* transition only, it gives absorption maximum at 125nm.

9. II. n ⇾ σ* transition: In this type saturated compounds containing atoms with unshared electron pairs are undergo n ⇾ σ* transition. It require less energy than the σ ⇾ σ* type. Most of the absorption peaks appearing below 200nm. In the presence of polar solvents the absorption maximum tend to shift shorter wavelength Ex: Water , ethanol.

10. III. n ⇾ π* & π ⇾ π* transition Most organic compounds are undergo transitions for n ⇾ π* and π ⇾ π* transition. Because energies required for processes bring the absorption peaks into spectral region. Both transition require the presence of an unsaturated functional group to the ´∏´ orbitals . Ex: For π ⇾ π* ⧐ Alkenes compounds, alkynes carbonyl For n ⇾ π* ⧐ carbonyl compounds.

11. Beer´s law Beer´s law states that The intensity of a beam of monochromatic light decreases exponentially with increase in the concentration of absorbing species arithmetically. Accordingly. - dI /dc α I [The decrease in the intensity of incident light with conc. is proportional to intensity of incident light] - dI /dc = k I ... [ Removing and introducing constant of proportionality k ]

12. - dI / I = k d c .... [ rearranging terms ] - InI = kc + b ....... Eq 1 [ When conc =0 there is no absorbance hence I = I sub.in Eq 1. ] -In I = k×0+b -In I 0= b ...substituting the value in Eq 1 - InI = kc – In I In I - InI = kc In I /I= kc ....since ( logA -log B = log A/B)

13. I /I= e kc ... [ removing natural logarithm] I/ I 0 = e - kc ...[making inverse on both side ] I = I 0 e - kc ...[ Eq 2 For Beer´s Law ]

14. Lambert s law The rate of decrease of intensity (monochromatic light) with the thickness of the medium is directly proportional to the intensity of light. - dI / dt α I [This eq. Can be simplified similar to eq. 2 to get the following eq. By replacing c with t] I = I 0 e - kt .... Eq.3 Eq .2 & Eq.3 get combined I = I 0 e - kct

15. I = I 0 10 -kct ... ( convert. Nat. Log.to base 10 & k k×0.4343) I/ I 0 = 10 -kct ... (rearranging terms) I 0/I = 10 kct .....(inverse on both side) log I 0/I = kct ....( taking log on both side) Eq .4 These Eq. Infer that, A = kct ....since log I 0/I =A Insted of k we can use Є A = Є ct (Mathematically Eq.for Beer Lambert Law) Where A = Absorbance Є = molecular extinction coefficint

16. C= concentration of drug ( mmol /lit) T= path length.

17. Instrumentation Instruments for measuring the absorption of U.V. or visible radiation are made up of the following components; 1. Sources (UV and visible) 2. filter or monochromator 3. Sample containers or sample cells 4. Detector

18. Dia : diagramatic representation of u.v spectroscopy .

19. 1. Radiation source It is important that the power of the radiation source does not change abruptly over its wavelength range. The electrical excitation of deuterium or hydrogen at low pressure produces a continuous UV spectrum. The mechanism for this involves formation of an excited molecular species, which breaks up to give two atomic species and an ultraviolet photon Both Deuterium and Hydrogen lamps emit radiation in the range 160 - 375 nm. Quartz windows must be used in these lamps, and quartz cuvettes must be used, because glass absorbs radiation of wavelengths less than 350 nm.

20. Various UV radiation sources are as follows a. Deuterium lamp b. Hydrogen lamp c. Tungsten lamp  d. Xenon discharge lamp e. Mercury arc lamp Various Visible radiation sources are as follows a. Tungsten lamp b. Mercury vapour lamp c. Carbonone lamp

21. 2. filters or monochromators   All monochromators contain the following component parts; • An entrance slit • A collimating lens • A dispersing device (a prism or a grating) • A focusing lens • An exit slit Polychromatic radiation (radiation of more than one wavelength) enters the monochromator through the entrance slit. The beam is collimated, and then strikes the dispersing element at an angle.

22. The beam is split into its component wavelengths by the grating or prism. By moving the dispersing element or the exit slit, radiation of only a particular wavelength leaves the monochromator through the exit slit. 3. sample containers or sample cells A variety of sample cells available for UV region. The choice of sample cell is based on a) the path length, shape, size b) the transmission characteristics at the desired wavelength c) the relative expense

23. The cell holding the sample should be transparent to the wavelength region to be recorded. Quartz or fused silica cuvettes are required for spectroscopy in the UV region. Silicate glasses can be used for the manufacture of cuvettes for use between 350 and 2000 nm.The thickness of the cell is generally 1 cm. cells may be rectangular in shape or cylindrical with flat ends. 4. Detectors In order to detect radiation, three types of photosensitive devices are  a. photovoltaic cells or barrier- layer cell

24. b. phototubes or photoemissive tubes c. photomultiplier tubes Phototubes   are also known as photoemissive cells. A phototube consists of an evacuated glass bulb. There is light sensitive cathode inside it. The inner surface of cathode is coated with light sensitive layer such as potassium oxide and silver oxide. When radiation is incident upon a cathode, photoelectrons are emitted. These are collected by an anode. Then these are returned via external circuit. And by this process current is amplified and recorded .

25. CHOICE OF SOLVENTS It should not itself absorb radiations in the region under investigations. It should be less polar so that it has minimum interaction with the solute molecule. Most commonly: 95%ethanol Cheap, good dissolving power, does not absorb radiation above 210nm. Typical examples

26. SOLVENT EFFECT The position and intensity of an absorption band may shift when the spectrum is recorded in different solvents. A dilute sample solution is prefered for analysis. Most commonly used solvent is 95% ethanol. It is best solvent as it is cheap,transparent down to 210nm. Position as well as intensity of absorption maxima get shifted for a particular chromophore by changing the polarity of solvent. By increasing polarity of solvent→dienes .

27. Applications. Detection of functional groups. Detection of impurities. Qualitative analysis. Quantitative analysis.

28. Thank you .