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UV SpectroscopyLecture 81The energy is sufficient to promote or excite a molecular electron to a higher energy orbital. Consequently, sometimes called "electronic spectroscopy". The three types of electron transitions that give rise to UV or visible are : sigma ; pi and nonbonding n orbitals (those with unshared pairs of electrons). As a rule, energetically favored electron promotion will be from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO), and the resulting species is called an excited state.

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3When sample molecules are exposed to light having an energy that matches a possible electronic transition within the molecule, some of the light energy will be absorbed by valence (outer) electrons. these electrons are promoted from their normal (ground) states to higher energy (excited) states (as the electron is promoted to a higher energy orbital. An optical spectrometer records the wavelengths at which absorption occurs, together with the degree of absorption at each wavelength. The resulting spectrum is presented as a graph of absorbance (A) versus wavelength.The most useful region of the UV spectrum is at wavelength longer than 200 nm.100- 200 nm * for isolated double bond and * for ordinary carbon-carbon bond.The useful transitions 200-400 nm are for compounds with conjugated double bonds and some n * and n * transitions

4Because the absorbance of a sample will be proportional to the number of absorbing molecules in the spectrometer light beam (e.g. their molar concentration in the sample tube), it is necessary to correct the absorbance value The corrected absorption value is called "molar absorptivity", and is particularly useful when comparing the spectra of different compounds and determining the relative strength of light absorbing functions (chromophores). Molar absorptivity () is defined as:Molar Absorptivity, = A / c l(where A= absorbance, c = sample concentration in moles/liter & l = length of light path through the sample in cm.)Beers Law. states that the light absorbed is proportional to the number of absorbing molecules ie to the concentration of absorbing molecules.

5A second law Lamberts law tells us that the fraction of radiation absorbed is independent of the intensity of the radiation. Combining these two laws gives the BeerLambert law:log10I0/I = lcIO = the intensity of the incident radiationI = the intensity of the transmitted radiation = the molar absorption coefficientl = the path length of the absorbing solution (cm)c = the concentration of the absorbing speciesin mol dm-36Two useful pieces of information are themolar absorption coefficient, and max which is the wavelength at which maximum absorption occurs.These two pieces of information are used to identify a substance besides other spectroscopic techniques. However, if and max are known for a compound the concentration of the solution can be calculated. This is the most common application.7Absorption of polyenes

Ethene contains a simple isolated carbon-carbon double bond, but the other two have conjugated double bonds. In these cases, there is delocalisation of the pi bonding orbitals over the whole molecule.

c = E = hE = (hc)/ E 1/ E = energy; c = speed of light; = wavelength; = frequency; h = Plancks constant8Less energy is required to promote pi electron of 1,3-butadiene than is needed to promote a pi electron of ethene. The reason is that the energy difference between the HOMO (highest occupied molecular orbital) and the LUMO for conjugated double bonds less than the energy difference for an isolated double bond.Because less energy is needed for pi excitation of 1,3-butadiene, this absorbs UV radiation for longer wavewlength than does ethylene.

9As more conjugated double bonds are added to a molecule, the energy required to the first excited state decreases.

Sufficient conjugation shifts the absorption to wavelengths that reach into visible region of the spectrum.

10Compound with sufficient conjugation is colored. Example lycopene, carotene

Violet: 400 - 420 nm

Indigo: 420 - 440 nm

Blue: 440 - 490 nm

Green: 490 - 570 nm

Yellow: 570 - 585 nm

Orange: 585 - 620 nm

Red: 620 - 780 nm 11Absorption by aromatic systemsBenzene and other aromatic compounds exhibit more complex spectra than can be explained by simple * transitions.A value 260 nm is often reported as the max for benzene because this is the position of strongest absorption above 200 nm.The absorption of UV radiation by aromatic compounds composed of fused benzene rings is shifted to longer wavelengths as the number of rings is increased because of increasing conjugation and greater resonance stabilization of the excited state. 12

13Absorption arising from transitions of n electronsCompounds that contain N, O, S P or one of the halogens all have unshared n electrons.If the structure contain no bonds, these n electrons can undergo only n * transitions.Because the n electrons are of higher energy than either or electrons, so less energy is required to promote an n electron and the transition occur at longer wavelengths than * transitions. 14The * orbital is of lower energy than the * orbital.Consequently, n * transitions require less energy than n * transitions.

15Sample should be dissolved in suitable solvent that does not itself absorb light in the region under investigation.Most commonly used solvent is 95% ethanol, 1,4 dioxane, and cyclohexaneSolution must be placed in suitable container that is transparent to light which is made of quartz or fused silica 16Important definitionsChromophore: a covalently unsaturated group responsible for the electronic absorption e.g. C=C, C=O, NO2Auxochrome: a saturated group with non bonded electron which when attached to a chromophore alter both the wavelength and the intensity of the absorption e.g. OH, NH2, ClBathochromic shift: the shift to longer wavelength due to the effect of substitution or solvent effect (red shift)

17FlavonoidsUV is major technique for the structural analysis of flavonoids why?Small amount of pure material is requiredThe amount of structural information gained from UV spectrum is considerably enhanced by the use of specific reagents (NaOMe, NaOAc, H3BO3, AlCl3 and AlCl3/HClReact with one or more functional groups on the flavonoid nucleusAddition of each of these reagents separately to an alcoholic solution induce shift in UVApplication of UV in natural product18

Band I is considered to be associated with the absorption due to the B-ring (cinnamoyl system) and band II with the absorption involving the A-ring, benzoyl system

191- significance of sodium acetateNaOAc addition to methanol Spectrum induce bathochromic shift of 10-20 nm in band II indicate the presence of free OH at C-7Change in band I indicates the presence of 4`-OH

20Boric acid induced shiftWhen boric acid is added to sodium acetate spectrum of the compound if it gives bathochromic shift 5 to 20 nm indicate the presence of ortho-dihydroxy groups No shift.............. No ortho-dihydroxy groups .

21Significance of AlCl3Reagent for placing OH at C5 or C3 or ortho dihydroxy groupAlCl3 complex with flavonoid has OH at C5 or C3 or orthodihydroxy

In this case bathochromic shift of 40-65 nm is observed in Band I

22AlCl3 has different stability to HCl , with the addition of 5-10 drops (10% HCl) on AlCl3 spectrum show

23Significance of Sodium methoxide shiftAddition of NaOMe to MeOH spectrum increase shift in band I by 80 nm (C-4`-OH)It is important to decide if OH at C-4` occupied by sugar or free or OCH324ClassBand IIBand IFlavone*250-280300-350Flavonol*250-265350-385Flavanone*270-295300-330Dihydroxy flavonol289324 (sh)isoflavone245-270300-340 (sh)Chalcon220-270 nm340-390 nmaurone220-270370-430 nm25

265-hydroxyflavone

275-hydroxyflavoneMeOH 268, 296 sh, 333AlCl3 290, 318 sh, 393AlCl3 +HCl 291, 319 sh, 394AlCl3 shift 40- 65 nm in band I indicate the presence of OH at 5 position

AlCl3 shift 40- 65 nm in band I indicate the presence of OH at 5 positionFlavone: 250- 280 II, 300- 350 IFlavonol: 250- 265 II, 350- 385 IFlavanone: 270- 295 II, 300- 330 I

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Apigenin 29Apigenin MeOH: 267, 295sh, 336NaoAc: 284, 301, 376AlCl3 : 276, 301 sh, 346, 384AlCl3 +HCl: 276, 299, 340, 381AlCl3 shift 40- 65 nm in band I indicate the presence of OH at 5 positionNaoMe: 275, 324, 392

NaOMe to MeOH spectrum increase shift in band I by 80 nm (C-4`-OH)NaoAc bathochromic shift of 10-20 nm in band II indicate the presence of free OH at C-7Change in band I indicates the presence of 4`-OHAlCl3 shift 40- 65 nm in band I indicate the presence of OH at 5 position30