UV Spectroscopy- Pharmaceutical Analysis

UV Spectroscopy- Pharmaceutical Analysis (Introduction, UV Spectra, Molecular orbitals, Electronic Transitions, Instrumentation, Sample Handling, Selection Rules, Absorption and Intensity shift, Application of UV, Problem related to UV spectroscopy for GPAT/ NIPER)

1. N I PE R 1 PHARMACAD

2. N I PE R Introduction Spectroscopy is the tool for study of atomic & molecular structure. UV/visible spectroscopy is also called electronic spectroscopy because the absorption spectra are a result of the behaviour of electrons in the target molecules. It deals with interaction of electronic radiation with matter involving the measurement & interpretation of the extension of absorption or emission of EMR by molecule. It provides information about electronic properties of molecules Most important consequence of such interaction is the energy is absorbed or emitted by the matter in discrete amount called as quanta. 2

3. N I PE R Introduction UV radiation starts at blue end of visible light (4000 Å) & ends at 2000 Å. It divided into two spectral region- Near UV region- 2000Å – 4000Å. Far UV region - below 2000 Å. UV-spectroscopy involved with electronic excitation. 3

4. N I PE R Introduction The difference in energy between molecular bonding, non-bonding and anti-bonding orbitals ranges from 125 – 650 kJ/mole This energy corresponds to EM radiation in the ultraviolet (UV) region, 200-400 nm, and visible (VIS) regions 400-800 nm of the spectrum 4

5. Characteristics of UV-Vis spectra of Organic Molecules Absorb mostly in UV, unless highly conjugated system Spectra are broad, usually to broad for qualitative identification purposes The most common detector for HPLC

6. Introduction    The Spectroscopic Process In UV spectroscopy, the sample is irradiated with the broad spectrum of the UV radiation If a particular electronic transition matches the energy of a certain band of UV, it will be absorbed The remaining UV light passes through the sample and is observed From this residual radiation a spectrum is obtained with “gaps” at these discrete energies – this is called an absorption spectrum   N I PE R  6

7. According to MO concept when molecule irradiated with UV-VIS r a di a t i o n t h e tr a n s f e r o f e l e c tr o n t a k es p l a c e fr o m H O M O l e v e l t o LUMO level (valency shell MO’s) . Du r i n g t h e s e e l e c t r o n tr a n s f e r s , t h e m o l e c u l e a b s o r b s e n e r gy a n d absorbed energy converted into UV-VIS peaks/ bands . T h e tr a n s f e r o f e l e c tr o n s fr o m H O M O t o LU M O ( B M O - A B M O) i s called electronic excitations or transitions . N I PE R Molecular Orbitals 7

8. Graphical Representation of MOs E n e rg y s    n Atomic orbital Atomic orbital  s Molecular orbitals Occupied levels Unoccupied levels N I PE R 8

9. N I PE R All possible excitations 1 ) 2 ) 3 ) 4 ) 5 ) 6 ) * * * * n n * * Types of MOs 9

10. Observed electronic transitions From the molecular orbital diagram, there are several possible electronic transitions that can occur, each of a different relative energy: E n e r gy s   s   n s s  n n s      s    a l k a n e s c a r b o n y l s unsaturated cmpds. O, N, S, halogens c a r b o n y l s N I PE R 10

11. Observed electronic transitions Special equipment to study vacuum or far UV is required Routine organic UV spectra are typically collected from 200-700 nm This limits the transitions that can be observed:  n n s s  s     s    carbonyls alkanes 150 nm carbonyls 170 nm unsaturated cmpds.180 nm √ - if conjugated! O, N, S, halogens 190 nm 3 nm √ E n e r gy 11 N I PE R

12. I n s tr u m e n t a t i on s am p l e re f e r e n c e de t e c t o r I I I I The construction of a traditional UV-VIS spectrometer is very similar to an IR, as similar functions – sample handling, irradiation, detection and output are required Here is a simple schematic that covers most modern UV spectrometers: log(I /I) = A 20 70  , nm monochromator/ beam splitter optics UV-VIS sources N I PE R 12

13. I n s tr u m e n t a t i on Two sources are required to scan the entire UV-VIS band: Deuterium lamp – covers the UV – 200 – 330 Tungsten lamp – covers 330 – 700 As with the dispersive IR, the lamps illuminate the entire band of UV or visible light; the monochromator (grating or prism) gradually changes the small bands of radiation sent to the beam splitter The beam splitter sends a separate band to a cell containing the sample solution and a reference solution The detector measures the difference between the transmitted light through the sample (I) vs. the incident light (I ) and sends this information to the recorder N I PE R 13

14. I n s tr u m e n t a t i on s am p l e Polychromator – entrance slit and dispersion device As with dispersive IR, time is required to cover the entire UV-VIS band due to the mechanism of changing wavelengths A recent improvement is the diode-array spectrophotometer - here a prism (dispersion device) breaks apart the full spectrum transmitted through the sample Each individual band of UV is detected by a individual diodes on a silicon wafer simultaneously – the obvious limitation is the size of the diode, so some loss of resolution over traditional instruments is observed Diode array UV-VIS sources N I PE R 14

15. I n s tr u m e n t a t i on Instrumentation – Sample Handling Virtually all UV spectra are recorded solution-phase Cells can be made of plastic, glass or quartz Only quartz is transparent in the full 200-700 nm range; plastic and glass are only suitable for visible spectra Concentration (we will cover shortly) is empirically determined A typical sample cell (commonly called a cuvet ): N I PE R 15

16. I n s tr u m e n t a t i on Instrumentation – Sample Handling Solvents must be transparent in the region to be observed; the wavelength where a solvent is no longer transparent is referred to as the cutoff Since spectra are only obtained up to 200 nm, solvents typically only need to lack conjugated π systems or carbonyls Common solvents and cutoffs: acetonitrile 19 chloroform 24 cyclohexane 19 5 1,4-dioxane 21 5 95% ethanol 20 5 n-hexane 20 1 methanol 20 5 isooctane 19 5 water 19 N I PE R 16

17. Instrumentation and Spectra Instrumentation – Sample Handling A dd iti o n al l y s o l v e n ts m u s t p r e s e r v e t h e f i n e s t r u c t u r e ( w h e r e it is actually observed in UV!) where possible H-bonding further complicates the effect of vibrational and rotational energy levels on electronic transitions . The more non-polar the solvent, the better (this is not always possible) N I PE R 17

18. Transitions are faster due to parallel arrangement of π and π* orbital's. I n t er a c t i o ns / o v erl a pping i s m o r e irrespective of energy gap. T r a n s i t i o n s s l o w du e t o p er p e nd i c u l a r a rr a n g e m e n t o f n and π* orbitals. These transitions may be due to v i b r a t i o n s o r t w i s t i n g o f t h e bonds. N I PE R Allowed excitations are more probable and faster Forbidden excitations (less probable) Electronic Excitations 18

19. *** Selection Rules Not all transitions that are possible are observed For an electron to transition, certain quantum mechanical constraints apply – these are called “ selection rules ” For example, an electron cannot change its spin quantum number during a transition – these are “ forbidden ” Other examples include: the number of electrons that can be excited at one time symmetry properties of the molecule symmetry of the electronic states To further complicate matters, “forbidden” transitions are sometimes observed (albeit at low intensity) due to other factors N I PE R 19

20. *** Selection Rules The symmetry allowed excitations are high probable excitations. Ex: π—π* Symmetry forbidden excitations are low probable excitations Ex: n—π* Ex ci t a t i o n s c a n t a k es p l a ce a m o n g B M O t o A B M O a n d N B M O t o ABMO’S. During electronic transition spin inversion is forbidden. N I PE R 20

21. 5) During electronic transitions change in multiplicity is forbidden. N I PE R *** Selection Rules 21

22. 6) Change in position of nuclei of bond during electronic transition is forbidden (Frank Condon Principle). (There is no change in the internuclear distance of the molecule during the excitation process. This is known as “Franck-Condon principle”.) N I PE R *** Selection Rules 22

23. Representation of UV spectrum diagram Each UV-band is characterized with its intensity and its position. Є or logЄ α Intensity. Forbidden transitions UV band are low intense bands. Є <100 Allowed transitions UV bands are high intense bands Є>10000 (10,000, 50,000, 100,000) N I PE R 23

24. Є = Molar absorptivity or Molar extension coefficient From Beer’s- Lamberts law A = Є. c. l Where A = Absorbance ( no units) c = concentration (moles/lit) l = length of solution or thickness of sol. or length of the tube/cell N I PE R Representation of UV spectrum diagram 24

25. Q: Certain sample solutions concentration is 0.001 gms/100 ml. M.Wt is 424, l = 1 cm, A = 0. 3025. Calculate Є ? N I PE R Problems 25

26. Q: For a solution of camphor in hexane in a 10 cm cell absorbance at 295 nm was found to be 2.52. What is the concentration of Camphor ? Molar extenction coefficient is 14. N I PE R Problems 26

27. Important Terms 200 to 800 nm a) Chromophore:- Any group which is absorbing energy from UV-VIS range of radiation called as Chromophore. b) Auxochrome:- The group which can not absorb radiation 200 to 800 nm range is the auxochrome. Auxochrome shift UV band towards higher λ side (right). It is called Bathochromic shift . Ex:- lone pair electron groups and –Ve charged groups. N I PE R 27

28. c) Bathochromic Shift:- Movement of UV pe a k t o w a rds hi g h e r λ s ide ( ri g ht s ide ) is c a lled t he Bathochromic shift (red shift). d) Hypsochromic shift:- Movement of UV pe a k o r b a nd t o w a rds l o w e r λ s ide ( le f t s ide) is t he Hypsochromic shift (Blue Shift). e) Hyperchromic shift :- Increase in intensity of UV peak is hyperchromic shift (more Є or logЄ value) f) Hypochromic shift:- Decrease in intensity of UV peak of band (lower Є or logЄ value). N I PE R Important Terms 28

29. Absorption & Intensity Shifts N I PE R 29

30. 30 Theoretical prediction of λ max of conjugated π-system ***Woodward – fieser rules (Empirical rules) Eg: 1,3-butadiene system, c=c-c=c Parent acyclic diene 217 nm (base values, π-π*) Parent hetero annular diene 215 nm ( ,, ) Parent homo annular dienes 253 nm ( ,, ) Increments for the substituents Al k y l g r o u p o r ri n g r e s i du es + 5 F o r e a ch e x o c y clic d o ub le b o n d + 5 3) 4) 5) Double bond with extending conjugation +30 On diene if halogen present +5 On diene if alkoxy group present +6 N I PE R

31. Limitation:- These rules are valid for unsaturated system which is having less than four double bonds in conjugation. 1) Acyclic C=C-C=C 217 nm 2) Hetero annular (two double bonds not in the same ring) 3) Homo annular (two double bonds with conjugation in same ring system) 4) If in the same molecule homo and hetero dienes present, homo diene is the base value (higher value) N I PE R 31

32. Q: Calculate the Absorption Maximum? N I PE R 32

33. Q: Calculate the Absorption Maximum? N I PE R 33

34. Q: Calculate the Absorption Maximum? N I PE R 34

35. Applications of UV-Visible Spectroscopy 35

36. 36 Why should we learn this stuff? After all, nobody solves structures with UV any longer! Many organic molecules have chromophores that absorb UV UV absorbance is about 1000 x easier to detect per mole than NMR Still used in following reactions where the chromophore changes. Useful because timescale is so fast, and sensitivity so high. Kinetics, esp. in biochemistry, enzymology. Most quantitative Analytical chemistry in organic chemistry is conducted using HPLC with UV detectors One wavelength may not be the best for all compound in a mixture. Affects quantitative interpretation of HPLC peak heights

37. 37 Practical Applications Pharmacy Practice Ultraquin (psoriasis med. Needs UV. Act. Pregnancy tests (colorimetric assays) Blood glucose tests, Bilichek ELISA’s Pharmaceutics pH titrations, purity measurement concentration measurement

38. 38 Pharmaceutical Applications On Line Analysis of Vitamin A and Coloring Dyes for the Pharmaceutical Industry Determination of Urinary Total Protein Output Analysis of total barbiturates Comparison of two physical light blocking agents for sunscreen lotions Determination of acetylsalicylic acid in aspirin using Total Fluorescence Spectroscopy Automated determination of the uniformity of dosage in Quinine Sulfate tablets using a Fibre Optics Autosampler Determining Cytochrome P450 by UV-Vis Spectrophotometry Light Transmittance of Plastic Pharmaceutical Containers

39. Applications of UV-Visible Spectroscopy 39 Detection of functional groups Estimation of extent of conjugation Distinction in conjugated and non-conjugated compounds Identification of an unknown compound Examination of Polynuclear hydrocarbons Elucidation of the structure of Vitamins A and K Preference over two tautomeric forms Identification of compound in different solvents Determination of configurations of Geometrical isomers Distinguishes between Equatorial and Axial conformations Determination of strength of H-bonding Hindered rotation and conformational analysis

40. 40 UV vs. IR vs. NMR UV has broad peaks relative to IR & NMR UV has less information than IR & NMR UV spectra are easier to collect UV spectra are faster to collect UV spectrometers are cheaper UV spectra require only nanograms of material or chemicals

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