UV-Visible spectroscopy

introduction
principle
electronic transitions
beers-lamberts law
instrumentation
types of uv-visible spectroscopy
applications

1. UV-VISIBLE SPECTROSCOPY MODERN PHARMACEUTICAL ANALYTICAL TECHIQUES Pharmaceutical Analysis LIKITHA BOGA M.PHARMACY I-I

2. INTRODUCTION Spectroscopy is the branch of science that deals with the study of interaction of electromagnetic radiation with matter. Atomic Spectroscopy: This Spectroscopy is concerned with the interaction of electromagnetic radiation with atoms. Molecular Spectroscopy: This Spectroscopy deals with the interaction of electromagnetic radiation with molecule.

3. Frequency (ν): It is defined as the number of time electrical field radiation oscillates in one second. unit for frequency is Hertz (Hz). Wavelength (λ): It is the distance between two nearest parts of the wave in the same phase i.e. distance between two nearest crest or troughs.

4. PRINCIPLE It is the measurement and interpretation of Electromagnetic radiation absorbed or emitted when the molecules or atoms or ions of sample move from one energy state to another. Electromagnetic radiation is given by: E = hν Where, E = energy (in joules) h = Planck’s constant (6.62 × 10 -34 Js) ν = frequency (in seconds)

5. UV SPECTROSCOPY UV spectroscopy is concerned with the study of absorption of UV radiation which ranges from 200nm to 400nm, colored compounds absorb the radiation from 400nm to 800nm(visible region). Colorless compounds absorb the radiation at UV region. In both UV spectroscopy and visible spectroscopy, the valence electrons absorb energy and there by molecules undergo transition from ground state to excited state. This absorption is characteristic and depends on nature of electrons present in the valence shell of the compound.

6. ELECTRONIC TRANSITIONS The absorption of light by a sample in the ultraviolet or visible region is accompanied by a change in the electronic state of the molecules in the sample. The energy supplied by the light will promote electrons from their ground state orbital to higher energy or excited state orbital or anti-bonding orbital. Any molecule has either n, π or σ or combination of these electrons .

7. σ- σ*transitions: σ electron from orbital is excited to corresponding anti-bonding orbital σ*.The energy required is large for this transition.  Example: Methane (CH 4 ) has C-H bond only and can undergo σ → σ* transition and shows absorbance maxima at 125nm. π-π* transitions: π electron in a bonding orbital is excited to corresponding anti-bonding orbital π*. Compounds containing multiple bonds like alkenes, alkynes, carbonyl, nitriles , aromatic compounds, etc undergo π → π* transitions. Example: Alkenes generally absorb in the region 170 to 205nm.

8. n- σ* transitions: Saturated compounds containing atoms with lone pair of electrons like O, N, S and halogens are capable of n → σ* transition. These transitions usually requires less energy than σ → σ* transitions. The number of organic functional groups with n→ σ* peaks in UV region is small (150 – 250nm). n - π* transitions: An electron from non-bonding orbital is promoted to anti-bonding π* orbital. Compounds containing double bond involving hetero atoms (C=O, C≡N, N=O) undergo such transitions. n → π* transitions require minimum energy and show absorption at longer wavelength around 300nm.

9. ELECTRONIC TRANSITION A transition in which a bonding σ electron is excited to an anti-bonding σ orbital is referred to as σ to σ* transition. In the same way π to π* represents the transition of one electron of a lone pair (non- bonding electron pair) to an anti-bonding π orbital. σ-σ* > n- σ* > n- π* > π-π*

10. LAWS Beer’s law: This law states that, “ the amount of light absorbed by a material is proportional to the concentration ”. Lambert’s law: This law is states that “ The amount of the light absorbed is proportional to the thickness of the absorbing material & is independent of the intensity of the incident light ”.

11. Beer-Lambert’s law This combined law states that the amount of light absorbed is proportional to the Concentration of the absorbing substance & to the thickness of the absorbing material. A = ε b c A = absorbance ε = molar absorbtivity with units of L /mol.cm b = path length of the sample ( cuvette ) c = Concentration of the compound in solution, expressed in mol /L

12. INSTRUMENTATION Components of UV-Vis Spectrophotometer: Light Source Filters and Monochromators Sample cell Detectors Recording Device

13. LIGTH SOURCE Ideal Characteristics of a Light Source: a. It should be stable and should not show fluctuation. b. It should provide light of sufficient intensity. c. It should be economical. d. It should emit a continuous spectrum. e. It should be simple in construction and operation.

14. TYPES OF LIGHT SOURCE a. Hydrogen Discharge Lamp b. Deuterium Lamp c. Xenon Arc Lamp d. Tungsten Halogen Lamp HYDROGEN DISCHARGE LAMP : In Hydrogen discharge lamp pair of electrodes is enclosed in a glass tube filled with hydrogen gas. When current is passed through these electrodes maintained at high voltage, discharge of electrons occurs which excites hydrogen molecules which in turn cause emission of UV radiation.

15. DEUTERIUM LAMP: It is similar to Hydrogen discharge lamp but instead of Hydrogen gas, Deuterium gas is used. It provides radiation in the range ( 185 - 380nm). The spectroscopic technique is not useful below 200nm since oxygen absorbs strongly at 185nm.The region below 200nm is called vacuum UV-region.

16. XENON ARC LAMP: In this xenon gas is stored under pressure. The UV- light produced by this lamp is of a greater intensity compared to hydrogen discharge lamp. Since the lamp operates at a high voltage, it becomes very hot during operations and hence needs thermal insulation. Emission of visible radiation also occurs along with the UV-radiation. Wavelength range (200 – 1000)nm.

17. TUNGSTEN HALOGEN LAMP: It is a special class of lamp with iodine added to the normal filling gas. The envelope is made up of quartz to tolerate higher lamp operating temperatures. Often a heat absorbing filter is inserted between the lamp and the sample holder to remove IR-radiations. The glass envelope absorbs strongly below 350nm. Wavelength range (350 – 3000)nm.

18. MONOCHROMATORS A monochromator is a device which converts a polychromatic light to monochromatic light. Types of monochromators : Prism monochromators : They are usually made up of glass, quartz or fused silica. Refractive type Reflective type

19. Grating monochromators : Gratings are made up of glass, quartz or alkyl halides like KBr and NaBr . Back surface of the gratings are coated with aluminium to make them reflective. Two types of gratings Diffraction gratings Transmission gratings Diffraction gratings: It works on the mechanism of reinforcement(strengthening). The incident rays are reinforced with those reflected, resulting in radiation whose wavelength is expressed by equation.

20. λ= d( sin i ± sin r ) n Where, n = order number (0,1,2,3) λ = wavelength of the resultant radiation d = grating spacing i = angle of incidence r = angle of reflection

21. Transmission gratings: In this type of grating the refracted rays produce reinforcement. When the transmitted radiations reinforce with the refracted radiations, a resultant radiation is obtained whose wavelength is given by the equation. λ= d( sin ø ) n λ = wavelength of the resultant radiation d= grating spacing Ø= angle of diffraction n= order number(0,1,2,3)

22. FILTERS A filter is a device which allows only the light of required wavelength to pass through and absorbs the unwanted radiation. Types of filters: Absorption filters Interference filters

23. SAMPLE CELL Sample cells or cuvettes are used to hold the sample solutions. Some typical materials are: Optical Glass - 335 - 2500nm Special Optical Glass – 320 - 2500nm Quartz (Infrared) – 220 - 3800nm Quartz (Far-UV) – 170 - 2700nm

24. DETECTORS Detectors are the devices which convert light energy into electrical signals that are displayed on the read out device. Sample absorbs a part of radiation and the remaining is transmitted. The transmitted radiation falls on the detector which determines the intensity of the radiation. Types of detectors: Photo multiplier tube Photovoltaic cell Photo tubes

25. Photo multiplier tube: It works on the principle of multiplication of the photo electrons by secondary emission of electrons. The emission of electrons is increased by a factor of 4 or 5 due to secondary emission of electrons.

26. Photo voltaic cell: When light rays falls on the selenium layer electrons are generated and taken by the photocathode. Electrons get accumulated which results in the generation of electric current. The current flow causes deflection in the galvanometer which gives the measure of the intensity of the radiation.

27. Phototubes: When lights falls on the photocathode electrons are produced that travel towards the collector anode and generate current. The amount of current generated is directly proportional to the intensity of light.

28. TYPES OF UV SPECTROPHOTOMETERS SINGLE BEAM UV VISIBLE SPECTROPHOTOMETER:

29. DOUBLE BEAM UV VISIBLE SPECTROPHOTOMETER:

30. APPLICATIONS Detection of Impurities- UV absorption spectroscopy is one of the best methods for determination of impurities in organic molecules. Structure elucidation of organic compounds- UV spectroscopy is useful in the structure elucidation of organic molecules, the presence or absence of unsaturation , the presence of hetero atoms. Quantitative analysis- UV absorption spectroscopy can be used for the quantitative determination of compounds that absorb UV radiation. Qualitative analysis- UV absorption spectroscopy can characterize those types of compounds which absorbs UV radiation. Identification is done by comparing the absorption spectrum with the spectra of known compounds.

31. Detection of Impurities UV absorption spectroscopy is one of the best methods for determination of impurities in organic molecules. Additional peaks can be observed due to impurities in the sample and it can be compared with that of standard raw material. By also measuring the absorbance at specific wavelength, the impurities can be detected. Benzene appears as a common impurity in cyclohexane . Its presence can be easily detected by its absorption at 255nm.

32. CHOICE OF SOLVETS A solvent is a liquid that dissolves another solid, liquid or gaseous solute resulting in a solution at specified temperature. Solvents can be broadly classified into two categories: i ) Polar ii) Non polar A drug may show varied spectrum at particular wavelength in one particular condition but shall absorb partially at the same wavelength in another conditions. The changes in the spectrum are due to Nature of solvent Nature of absorption band Nature of solute

33. Nature of solvent: most commonly used solvent is 95% ethanol. It is best solvent as: It is cheap. Has good dissolving power. Does not absorbs radiations above 210nm. In choosing a solvent, consideration must be given not only to its transparency, but also to its possible effects on absorbing system. Benzene, chloroform, carbon tetrachloride can’t be used because they absorb in the range of 240-280nm. It should not itself absorb radiations in the region under investigation. It should be less polar so that it has minimum interaction with the solute molecules.

34. Common solvents used in UV spectra: SOLVENT WAVELENGTH(nm) Water 205 Methanol 210 Ethanol 210 Ether 210 Cyclohexane 210 Dichloroethane 220

35. EFFECT OF SOLVENT A solvent exerts a profound influence on the quality and shape of spectrum. The absorption spectrum of pharmaceutical substance depends practically upon the solvent that has to been employed to solubilize the substance. A drug may absorb a maximum radiation energy at particular wavelength in one solvent but shall absorb partially at the same wavelength in another solvent. Example: Acetone in n-hexane λ max at 279nm. Acetone in water λ max at 264nm.

36. DIFFERENCE SPECTROSCOPY The selectivity and accuracy of spectrophotometric analysis of sample containing absorbing interference may be markedly Improved by the technique of difference spectrophotometry . Advantages: The selectivity and accuracy of spectrophotometric analysis of samples containing absorbing interferents may be markedly improved by the technique of difference spectrophotometry . A substance whose spectrum is unaffected by changes of pH can be determined by difference spectrophotometric procedures.

37. DERIVATIVE SPECTROSCOPY Another simplest method for an increasing a selectivity is derivatization of spectra. this operation allows to remove spectral interferences and as a consequence leads to increase selectivity of assay. It involves the conversion of a normal spectrum to it’s first, second or higher derivative spectrum. The normal absorption spectrum is referred to as the fundamental zero order or D spectrum. The first derivative D 1 spectrum is a plot of the rate of change of absorbance with wavelength against wavelength dA / dʎ .

38. Multi -component analysis: Derivative spectrophotometry (DS) has been mainly used in pharmaceutical analysis for assaying of a main ingredient in a presence of others components or its degradation product. Calculation of some physico -chemical constants, e.g. reaction, complexation or binding constants. The main disadvantage of derivative spectrophotometry is its poor reproducibility.

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