Applications of uv spectroscopy, by Dr. Umesh Kumar Sharma and Anu Mathew

Applications of uv spectroscopy, by Dr. Umesh Kumar Sharma and Anu Mathew.

1. APPLICATIONS OF UV- VISIBLE SPECTROSCOPY 1 By: Dr. Umesh Kumar Sharma & Anu Mathew Department Of Pharmaceutics, Mar Dioscorus College Of Pharmacy, Alathara , Sreekaryam , Thiruvananthapuram

2. 2 Absorption of light in the UV / visible part of the spectrum. Transitions that result in absorption of the electromagnetic radiation in this region. The most probable transition from highest occupied molecular orbital to lower occupied molecular orbital. UV spectroscopy is routinely used in analytical chemistry for quantitative determination of different analytics such as transition metal ions, highly conjugated organic compounds and biological macromolecules .

3. Qualitative applications 3 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. UV absorption spectroscopy is generally used for characterizing the aromatic compounds & conjugated olefins.

4. 4 Detection of conjugation . Conjugation may be between, Between two/more C=C bonds or C≡C bonds. Between C=C or C=O bonds, Between double bonds and aromatic ring. Presence of an aromatic ring & the number & the locations of substituents attached to the carbons of the conjugated system. Eg : No. of bonds λ max Ethylene (CH 2 =CH 2 ) 1 174nm A cetone (CH3-CO-CH3) 2 217nm

5. 5 Structure of inorganic complexes Used to distinguish between cis and trans isomers. Geometrical isomerism-easily distinguished from visible spectra Eg : [Co(en) 2 F 2 ]NO 3 : Cis form - violet color, Trans form - green color. Planar tetrahedral equilibrium * Planar form- as temp↑ intensity of spectra ↓ . * Tetrahedral form- as temp ↑ intensity ↑ . Equilibrium shift from planar to tetrahedral complex as ↑ temp. Eg : Schiff base

6. 6 Detection of functional groups Detect the presence or absence of a functional group in a compound. Absence of a band at particular wavelength regarded as an evidence for absence of particular group. a) λ max for CH 3 -COOH 208nm CH 3 -CO-CH 3 189nm b) Carbonyl group absorption band between 280-290nm Aromatic ring absorption band at 260nm.

7. 7 Detection of impurities The bands due to impurities are very intense The organic compounds can be classified into saturated compounds having little absorption & unsaturated compounds having strong absorption bands. Eg : The common impurity in cyclohexane is benzene. Its presence can be easily detected by its absorption at 255nm.

8. 8 Determination of pKa value of indicator acid or base : Let us consider an acid HA. It undergoes dissociation in water to form H 3 O + & A -. HA + H 2 O H 3 O + + A - A- conjugated base. The acid dissociation Ka is defined by the expression Ka =[H 3 O] + [A ] [HA ] On taking logarithm on both sides - log Ka = log [H 3 O + ]-log [A - ]/[HA] pKa = pH –log [A]/[HA] pKa = pH+ log [HA]/[A ] The ratio [HA]/[A] can be determined by spectrophotometer from the graph plotted between absorbance & wavelength at different pH values.

9. 9 Quantitative applications Used to determine quantity of compounds Used in Pharmaceutical research, chemical research, biochemistry, chemical analysis & industrial processing Measurement of unknown concentration by : Calibration curve method The solution of most of the drug obey beer-lambert’s law up-to certain limit By making serial solution from a stock solution & calibration graph is drawn, concentration along x axis and absorbance along y axis. Eg : beer-lambert’s limit for cetirizine is 2-16 μ g/ml

10. 10 b) Sensitivity UV absorption analysis is frequently quite sensitive. It determines compounds of concentration >1ppm The sensitivity determines the lowest concentration & that can be determined quantitatively by this method. Molecular weight determination Can be measured spectrophotometicaly by preparing the suitable derivative of these compounds. Eg : To determine the molecular weight of amine, it is converted to amine picrate. Then known concentration of amine picrate is dissolved in a liter of solution & its optical density is measured at 380nm .

11. 11 Charge transfer transitions Eg : when iodine & benzene are brought together in a 1:1 mole ratio in heptane, a new absorption band is observed, which is not observable in the spectra of individual components i.e. benzene & iodine. According to mulliken , this new band arises due to the absorption of radiation by a molecular complex formed between benzene & iodine. This can be represented as D+A ( DA)complex where D is the benzene. DA D + A - (covalent bond) (ionic bond) The total wave function of the ground state may be put as Ψ = Ψ(DA)+ λ Ψ(D + A - ) λ = measure of charge transfer

12. 12 Tautomeric equilibrium Determine the percentage of various keto & enol forms present in a tautomeric equilibrium . Eg : ethyl acetoacetate CH 3 COCH 2 COOCH 5 CH 3 C(OH)=CHCOOC 2 H 5 The keto form has λ max 275nm & ε =16. this has only the weak n→ Π * band of the isolated carbonyl group. The enol form has λ max 244nm & ε =16000. from the strength of the 244nm band can measure the proportions of tautomers present in the ethyl acetoacetate.

13. 13 Structure of chloral T his compound can have any one of the following structures H CCl 3 -C-OH CCl 3 -CHO OH (I) (II) When the UV absorption spectrum of this compound is recorded in hexane, it shows a band at 290nm. On the other hand the UV spectrum of chloral in aqueous solution does not show any band. This confirms that chloral hydrate has structure I rather than II.

14. 14 Chemical kinetics In order to determine the kinetics of a reaction the change in the concentration of either a reactant or a product with time is measured. Based on the fact that one of the reactants / products exhibiting suitable absorption in the UV region is not overlapped by absorption due to other species present. In this method , two solutions are entering through X & Y. Then, these are allowed to pass through reaction chamber B & the flow of the mixed solutions is stoppered by piston D. The absorbance of any species which absorbs in the UV region is measured at C with a UV spectrophotometer. Photomultipier is used as a detector whose output is displayed on the screen with a time base .

15. 15 Multicomponent analysis A bsorbance of the sample is the sum of the absorbance of the individual components. The selectivity and accuracy of spectrophotometric analysis of samples containing interfering substances can be improved by derivative and difference spectroscopy. Derivative spectroscopy The change in absorbance with respect to wavelength is recorded. 1 st and 2 nd derivative spectrum is recorded & characteristic peak for individual components can be identified & quantified, using caliberation curve of pure substance . Difference spectroscopy Useful to quantify a substance when interfering species are present. Absorbance difference between 2 forms of same drug is measured. Achieved by using pH manipulation using a pair of buffers. Used to quantify drugs in biological fluids .

16. 16 Photometric titrations : Plot of absorbance as a function of volume of titrant Usual titrimetric disadvantages can be overcome by spectrophotometric titrations using spectrophotometer which determines the end point. Method : Titration vessel is kept directly in light path of the instrument Titrant added Absorbance measured Plot of absorbance v/s volume of titrant

17. 17 If titration reaction completes, the titration curve shows 2 straight lines intersecting at equivalence point. If reaction is incomplete gives a curvature at equivalence point region, extrapolation of two line segments of titration curve gives equivalence point. Characteristic of a case where only titrant absorbs : Eg : Titration of A rsenic (As) with bromate - bromide mixture. Absorbance are taken at wavelength were bromine absorbs. At the initial period, absorbance will not be changed since only As remains in solution. As titration starts and titrant added ,volume increases no change in absorbance. After completion of As, bromine alone in vessel, increases the absorbance due to its color . The extrapolation of 2 lines gives the equivalence point.

18. 18 b) Only product of the reaction absorbs : Eg : Titration of Cu I with EDTA of 745nm. This wavelength is selected because at this position only EDTA –Cu complex possess greater absorbance compared to copper solution alone. c) Only the analyte absorbs and product do not absorb : Eg : Titration of p-toluidine with per-chloric acid at 290nm. This wavelength is selected because p-toluidine absorbs at this wavelength where as perchloric acid has no absorbance in that region. As soon as whole qty. of p -toluidine has reacted, the absorbance will become constant at equivalence point. d ) When a colored analyte is converted into colorless compound by a colored titrant . W hen titrant is added, color of analyte starts fading & form colorless product. After equivalence point absorbance again rises due to the color of the titrant alone. a.

19. 19 . a. Characteristic of a case where only titrant absorbs b) Only product of the reaction absorbs c) Only the analyte absorbs and product do not absorb d) When a colored analyte is converted into colorless compound by a colored titrant .

20. 20 References Instrumental methods of chemical analysis by Gurudeep R Chatwal , Page no. 2.177 - 2.182. Principles of instrumental analysis, by Skoog , Page no. 375-379. Organic spectroscopy by William kemp , Page no. 261-267. Pharmatutor.com wikipedia

21. 21 THANK YOU For Feedback / Comments Write to [email protected]