UV-visible spectroscopy - 2021
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ELECTROMAGNETIC RADIATION THEORY ABOUT UV-VISIBLE SPECTROSCOPY INSTRUMENT FACTORS AFFECTING ABSORBTION Woodward-Fieser Rules APPLICATIONS
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UV-visible spectroscopy - 2021
- 1. Ph.D.Ahmed Metwaly Ultraviolet/visible spectroscopy ORCID account Email: ametwaly@azhar.edu.eg 1 • Associate Professor of Pharmacognosy , faculty of Pharmacy, Al-Azhar University • Senior research fellow, Liaoning University of Traditional Chinese Medicine, China (20118-2019) • Visiting scholar, School of Pharmacy, University of Mississippi, USA (2012-2014)
- 2. OBJECTIVES • ELECTROMAGNETIC RADIATION • THEORY ABOUT UV-VISIBLE SPECTROSCOPY • INSTRUMENT • FACTORS AFFECTING ABSORBTION • Woodward-Fieser Rules • APPLICATIONS 2
- 3. Electromagnetic Radiation Electromagnetic radiation consist of discrete packets of energy which are called as photons. A photon consists of an oscillating electric field (E) & an oscillating magnetic field (M) which are perpendicular to each other.
- 5. Electromagnetic Radiation Frequency (ν): It is defined as the number of times electrical field radiation oscillates in one second. The unit for frequency is Hertz (Hz). 1 Hz = 1 cycle per second 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.
- 6. Electromagnetic Radiation As photon is subjected to energy, so E = h ν = h c / λ
- 7. Electromagnetic Radiation The visible spectrum constitutes a small part of the total radiation spectrum. Most of the radiation that surrounds us cannot be seen, but can be detected by instruments.
- 8. Ultraviolet and Visible Spectroscopy: The ultraviolet and visible (UV/Vis) radiation is that region of EMR (electromagnetic radiation) or spectrum whose wavelengths range from 10-800 nm. The (UV/Vis) region can be divided into three types of radiation that are 1. Vacuum UV; λ = 10-200 nm 2. Near UV; λ = 200-400 nm 3. Visible radiation; λ = 400-800 nm
- 9. Electromagnetic Radiation Violet 400 - 420 nm Yellow 570 - 585 nm Indigo 420 - 440 nm Orange 585 - 620 nm Blue 440 - 490 nm Red 620 - 780 nm Green 490 - 570 nm
- 11. 11
- 12. The comon electronic transitions can graphically shown as:
- 14. Principles of Spectroscopy The 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). Spectrometer is an instrument design to measure the spectrum of a compound.
- 16. Instrumentation Components of UV-Visible spectrophotometer Source Filters & Monochromator Sample compartment Detector Recorder
- 18. Five Basic Optical Instrument Components 1) Source – A stable source of radiant energy at the desired wavelength (or range). 2) Wavelength Selector – A device that isolates a restricted region of the EM spectrum used for measurement (monochromators, prisms & filters). 3) Sample Container – A transparent container used to hold the sample (cells, cuvettes, etc). 4) Detector/Photoelectric Transducer – Converts the radiant energy into a useable signal (usually electrical). 5) Signal Processor & Readout – Amplifies or attenuates the transduced signal and sends it to a readout device as a meter, digital readout, chart recorder, computer, etc.
- 24. LIGHT SOURCES 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 follow a. Tungsten lamp b. Mercury vapour lamp c. Carbonone lamp SAMPLE COMPARTMENT
- 25. SUMMARY Types of source, sample holder and detector for various EM region REGION SOURCE SAMPLE HOLDER DETECTOR Ultraviolet Deuterium lamp Quartz/Fused silica Phototube, PM tube, diode array Visible Tungsten lamp Glass/Quartz Phototube, PM tube, diode array
- 26. Beer Lamberts Law: Beer Lamberts Law: 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
- 28. Chromophore Chromophore: covalently unsaturated groups responsible for electronic absorption e.g. NO2, N=O, C=O, C=N, C≡N, C=C, C=S, etc Auxochrome The functional groups attached to a chromophore which modifies the ability of the chromophore to absorb light , altering the wavelength or intensity of absorption. OR The functional group with non-bonding electrons that does not absorb radiation in near UV region but when attached to a chromophore alters the wavelength & intensity of absorption.
- 29. Factors affecting UV/Vis absorption: The position and intensity of an absorption band of a chromophore may be modified by substituent groups attached to the chromophore, type of solvent, degree of conjugation and stereochemical effect. Bathochromic (Red) shift: The shift of absorption to a longer wavelength. Hypsochromic ( Blue) shift: The shift of absorption to a shorter wavelength. Hyperchromic effect: The increase in absorption intensity. Hypochromic effect: The decrease in absorption intensity.
- 30. Application of UV/vis spectroscopy: Chrysophanol, λ max (EtOH) nm: 224, 254, 288, 432, By addition of one drop of 5% NaOH, a bathochromic shift will occurs; 232, 250, 330, 500. Ferulic acid, λ max (MeOH) nm: 230, 279. Vanillic acid, λ max (MeOH) nm: 252, 291, 328. Iridoids and secoiridoids, λ max (MeOH) nm: 236; characteristics for enol-ether system conjugated with carbonyl group (-OCO-C=CH-O-). UV Aspect of Flavonoids:
- 31. • When absorption maxima (λmax) of a compound shifts to longer wavelength, it is known as bathochromic shift or red shift. • The effect is due to presence of an auxochrome or by the change of solvent. • e.g. An auxochrome group like –OH, -OCH3 causes absorption of compound at longer wavelength. • Bathochromic Shift (Red Shift) 1
- 32. • In alkaline medium, p-nitrophenol shows red shift. Because negatively charged oxygen delocalizes more effectively than the unshared pair of electron. p-nitrophenol λmax = 255 nm λmax = 265 nm • Bathochromic Shift (Red Shift) 1 OH N + O - O OH - Alkaline medium O - N + O - O
- 33. • When absorption maxima (λmax) of a compound shifts to shorter wavelength, it is known as hypsochromic shift or blue shift. • The effect is due to presence of an group causes removal of conjugation or by the change of solvent. • Hypsochromic Shift (Blue Shift) 2
- 34. • Aniline shows blue shift in acidic medium, it loses conjugation. Aniline λmax = 280 nm λmax = 265 nm • Hypsochromic Shift (Blue Shift) 2 NH2 H + Acidic medium NH3 + Cl -
- 35. • When absorption intensity (ε) of a compound is increased, it is known as hyperchromic shift. • If auxochrome introduces to the compound, the intensity of absorption increases. Pyridine 2methylpyridine λmax = 257 nm λmax = 260 nm ε = 2750 ε = 3560 • Hyperchromic Effect 3 N N CH3
- 36. • When absorption intensity (ε) of a compound is decreased, it is known as hypochromic shift. Naphthalene 2-methyl naphthalene ε = 19000 ε = 10250 CH3 • Hypochromic Effect 4
- 37. Wavelength ( λ ) Absorbance ( A ) Shifts and Effects Hyperchromic shift Hypochromic shift Red shift Blue shift λmax
- 40. Woodward-Fieser Rules Email: ametwaly@azhar.edu.eg 40 1. Conjugated dienes 2. Conjugated dienones 3. Aromatic systems
- 41. Dienes Email: ametwaly@azhar.edu.eg 41 Acyclic butadiene = 217 nm
- 51. APPLICATIONS Email: ametwaly@azhar.edu.eg 51 •Detection of Impurities. ... •Structure elucidation of organic compounds. ... •Quantitative analysis. ... •Qualitative analysis. ... •HPLC •Chemical kinetics. ...
- 52. SUMMARY Email: ametwaly@azhar.edu.eg 52 • ELECTROMAGNETIC RADIATION • THEORY ABOUT UV-VISIBLE SPECTROSCOPY • INSTRUMENT • FACTORS AFFECTING ABSORBTION • Woodward-Fieser Rules • APPLICATIONS
- 53. Ph.D.Ahmed M.Metwaly Associate professor,Pharmacognosy department, Faculty of Pharmacy,Al-Azhar University. ametwaly@azhar.edu.eg Email: ametwaly@azhar.edu.eg 53