Ultraviolet and Visible Spectroscopy PDF

Ultraviolet and visible spectroscopy  Learning outcomes At the end of this session students shall be able to 1. Explain basic principles of UV-Visible spectroscopy 2. Explain relevant terms of UV-Visible spectroscopy 3. Explain working principle, taking spectra 4. Des...

Ultraviolet and visible spectroscopy  Learning outcomes At the end of this session students shall be able to 1. Explain basic principles of UV-Visible spectroscopy 2. Explain relevant terms of UV-Visible spectroscopy 3. Explain working principle, taking spectra 4. Describe the outline of UV spectroscopy device  Spectroscopy Spectroscopy is an analytical method that involve the interaction of molecules with electromagnetic energy Example of spectroscopic techniques are: Infrared (IR), ultraviolet-visible (UV-Vis) and nuclear magnetic resonance (NMR) spectroscopy.  Electromagnetic radiation (EMR) Electromagnetic radiation is a radiation consisted of discrete packages of energy called photons. The packages make the electromagnetic spectrum which is arbitrarily divided into regions  EMR Electromagnetic radiation has dual wave-particle behavior. As a wave it has: Wavelength, λ, = the distance btw successive trough or crest. Frequency, ν, = the number of cycles per second. The amplitude = the height of a wave, measured from midpoint to peak. The velocity, The Energy,  UV - Visible radiation The UV radiation extends from 10 – 400 nm 200 – 400 nm is near UV region below 200 nm is far UV region The visible radiation extends from 400–800 nm Note: Far UV spectroscopy is studied under vacuum condition  Principle of UV-Vis spectroscopy In UV-Vis radiation, a molecule absorb energy that promote an electron from a lower-energy orbital to a higher-energy one. E.g. In 1,3-butadiene absorption of UV radiation promotes an e- from HOMO to LUMO. (from ) (energy = 217 nm) HOMO = highest occupied molecular orbital LUMO = lowest unoccupied molecular orbital  Possible electronic transitions The UV-Vis spectroscopy is applicable to molecules capable of transitions (λ >200 nm) – Molecules containing conjugated  systems and lone pair of e-s.  The UV spectrum A UV spectrum is a chart that plots wavelength versus absorbance (A) UV/Vis absorption bands are broader because the transition btw electronic energy levels also include a transition between vibrational energy levels. Thus closely spaced absorption bands merge together to form a single broad band.  Terminologies: chromophore Chromophore: is the functional group capable of absorbing UV-Vis radiation E.g. C=C, C=S, C=O, C=N, NO2, N=O, C≡N 1. Non-conjugated alkenes 2. Non-conjugated C=O 3. Conjugated system 4. Conjugation of C=C and C=O shifts the of both groups to longer wavelength  Characteristics of some chromophores max max max max Chromophore Chromophore Chromophore Chromophore (nm) (nm) (nm) (nm) H2C CH2 171 178 227 255 Ethylene 1,5-hexadiene 1,3-hexadiene Benzene O O O O C 279 291 290 C OH 273 H3C CH3 H3C H Acetone Crotonaldehyde Benzoic acid Cyclohexanone O C C C OH 273 292 H H (CH2)3NHMe Cinnamic acid Protriptyline  Terminologies: Auxochrome Auxochrome: is the functional group attached to chromophore that alters the wavelength or intensity of absorption (e.g. –OH, -NH2, -CH3, -OCH3 ) Auxochrome modifies the ability of the chromophore to absorb light OH NH2 Benzene Phenol Aniline max= 255 nm max= 270 nm max= 280 nm  Absorption and intensity shifts Shift Description Example Bathochromic effect Shifts to longer wavelength Benzene λmax = 255 nm (Red shift) due to presence of an Phenol λmax = 270 nm auxochrome or change of p-nitrophenol (PNP) λmax = 255 nm solvent. PNP in alkaline medium λmax = 265 nm Hypsochromic effect Shift to shorter wavelength Aniline λmax = 280 nm (Blue shift) due to presence of groups Aniline in acid medium λmax = 265 nm that causes removal of conjugation or by the (loses conjugation) change of solvent Hyperchromic effect An increase in absorption Pyridine log ϵ = 3.3 2-picoline log ϵ = 3.4 Hypochromic effect A decrease in absorption Naphthalene log ε = 4.28 2-methyl naphthalene log ε = 4.01  Absorption and intensity shifts Hyperchromic shift Blue Red Absorbance ( A ) shift shift Hypochromic shift λmax Waveleng  Absorbance (A)/transmittance (T) When a solution of a sample is irradiated by EMR of intensity I0 where I = transmitted energy and I = incident energy 0 Absorbance But A  Beer–Lambert Law When a monochromatic radiation is passed through a solution, the decrease in the intensity of radiation with thickness of the solution is directly proportional to the intensity of the incident light and concentration of the solution gives Converting from ln to log, and substituting for absorbance, gives A = αbC where α is the analyte’s absorptivity with units of cm–1 conc–1. When concentration is expressed using molarity, the absorptivity is replaced by the molar absorptivity, ε (with units of cm–1 M–1) Thus A = εbC (Beer’s Law) The ε give the probability that the analyte will absorb a photon of given energy. Thus, the value for ε depend on the wavelength of electromagnetic radiation.  Beer’s Law - Quantitative Applications If ε and b are constants The Beer’s law gives A≈C Thus, solution of different concentrations of an analyte should give a linear calibration curve with no intercept. Accordingly, the calibration curve can be used to determine unknown concentration in sample of the same analyte  Beer’s law for pharmaceutical products To express concentrations in grams or milligrams the Beer’s law is written as follows for pharmaceutical products. where, A = absorbance A (1%, 1 cm) = absorbance of a 1% w/v solution in a 1 cm cell, l = pathlength in cm c = the concentration of the sample in g/100 ml (in g/ml) if l = 1cm  Examples What are the concentrations of the following solutions of drugs in g/100 ml and mg/100 ml? i. Carbimazole, A (1%, 1 cm) value = 557 at 291 nm, measured absorbance = 0.557 at 291 nm. ii. Hydrocortisone sodium phosphate, A (1%, 1 cm) value= 333 at 248 nm, measured Absorbance = 0.666 at 248 nm. iii. Isoprenaline, A (1%, 1 cm) value = 100 at 280 nm, measured absorbance, = 0.500 at 280 nm Answers: (i) Carbimazole 0.001 g/100 ml, 1 mg/100 ml (ii) Hydrocortisone sodium phosphate 0.002 g/100 ml, 2 mg/100 ml (iii) Isoprenaline 0.005 g/100 ml, 5 mg/100 ml.  A spectrophotometer Component Description Light Source lamps emitting UV-visible light. e.g. tungsten, deuterium and mercury lamp. Monochromator isolate individual wavelengths of light. e.g. prisms and Diffraction gratings Sample cell glass or Quartz (glass not suitable for UV region) Photodetectors convert transmitted radiation into electrical energy Analog to-digital (A/D) converters the signal to a voltage and digital.  Photodetectors Incident light Photocell on illumination cathode generates current that is Photodiode array (PDA) use proportional to incident pn-junction diode. less Photomultiplier tube (PMT) detects radiation. sensitive than PMT, but and amplifies radiant energy by Disadvantage: no excellent linearity, speed, dynodes, that produce a successively amplification. temperature and small size give them higher positive voltage. PM T is > sensitive , nonlinear at very advantage 200 times more sensitive than the low and very high phototube. illumination.  Double beam spectrophotometer Double-beam spectrophotometers permit automatic correction of sample and reference absorbance. The system performs continuous zeroing electronically Qualitative and Quantitative application  Examples of the Molecular UV/Vis Analysis of Clinical Samples Analyte Method λ(nm) total serum react with NaOH and Cu2+; forms blue-violet 540 protein complex serum cholesterol react with Fe3+ in presence of isopropanol, acetic 540 acid, and H2SO4; forms blue-violet complex uric acid react with phosphotungstic acid; forms tungsten 710 blue serum extract into CHCl3 to isolate from interferents and 260 barbiturates then extract into 0.45 M NaOH glucose react with o-toludine at 100oC; forms blue-green 630 complex protein-bound decompose protein to release iodide, which 420 iodine catalyzes redox reaction between Ce3+ and As3+; forms yellow colored Ce4+  Steroidal androgens and corticosteroids The chromophores of steroidal androgens and corticosteroids is enone. All show absorbance maxima of similar intensity, at around 240 nm. Shapes of the spectra may be different and can be used in qualitative identity tests. Steroid Mwt λmax A (1%, 1 cm) Hydrocortisone 362.5 240 435 Betamethasone 392.5 240 390 Clobetasol butyrate 479.0 236 330 Betamethasone 516.4 241 296 sodium phosphate  Ephedrine: the benzoid chromophore Ephedrine and other molecules having simple benzene ring as chromophore absorb at λmax = 260 nm [A (1%, 1 cm) = 12]. Like benzene its most intense absorption maximum is below 200 nm. its vibrational fine structure is preserved because the chromophore does not interact Similar drugs include: strongly with the solvent. diphenhydramine, amphetamine, ibuprofen and dextropropoxyphene.  Ketoprofen: extended benzene chromophore The chromophore in Ketoprofen is the extended benzoid system λmax = 262 nm, [A (1%, 1 cm) = 647, a bathochromic shift from the strong absorbance band in benzene (λmax = 204 nm). Similar drugs include: cyproheptadine, dimethindine, protriptyline, zimeldine.  Procaine: amino group auxochrome In procaine, the benzene chromophore is extended by the C=O and has an amino auxochrome all producing bathochromic shift under basic conditions, λmax = 279 nm with an A (1%, 1 cm) = 100. Under acidic conditions, λmax = 270 nm with an A (1%, 1 cm) = 1000 (a hyperchromic shift). Similar drugs include: procainamide and proxymetacaine.  Phenylephrine: hydroxyl group auxochrome The hydroxyl auxochrome interact with the chromophore under both acidic and alkaline conditions. Under acidic conditions λmax = 273 nm, A (1%, 1 cm) = 110 Under alkaline conditions λmax = 292 nm, A (1%, 1 cm) = 182 (bathochromic and hyperchromic shift)  Quantitative analysis Assay examples : Furosemide (frusemide) in tablet form Measured powder of furosemide (frusemide) is shaken with ~ 300 ml of 0.1 M NaOH to extract the acidic furosemide (frusemide). The extract is then made up to 500 ml with 0.1 M NaOH. A portion of the extract is filtered and 5 ml of the filtrate is made up to 250 ml with 0.1 M NaOH. The absorbance of the diluted extract is measured at 271 nm. A (1%, 1 cm) = 580 at 271 nm in basic solution. From the data below calculate the % of stated content in a sample of furosemide (frusemide) tablets: – Stated content per tablet: 40 mg of furosemide (frusemide) – Weight of 20 tablets: 1.656 g – Weight of tablet powder taken for assay: 0.5195 g – Absorbance reading: 0.596.