APPLICATION OF UV/VIS SPECTROSCOPY Dr Tahir Mahmood 1 APPLICATION OF UV/VIS SPECTROSCOPY CONTENTS: Detection of extent of conjugation Detection of functional groups Determination of the purity of a substance Identification of unknown compound Determination of configurations of geometrical isomers Quantitative analysis Dissociation constants of acids and bases 2 APPLICATION OF UV/VIS SPECTROSCOPY Detection of extent of conjugation • The extent of conjugation in the Dienes, Trienes and Polyenes can be detected with the help of UV spectroscopy. • The presence of conjugate double bond, decreases the energy difference between HOMO and LUMO of resulting diene. As the number of conjugated double bonds is increased, the gap between highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) is lowered. • Therefore, the increase in size of the conjugated system gradually shifts the absorption maximum (λmax) to longer wavelength and also increases the absorption. This figure shows the change in energy of MO on conjugation. 3 APPLICATION OF UV/VIS SPECTROSCOPY Detection of extent of conjugation Therefore, increase in size of the conjugated system gradually shifts the absorption maximum (λmax) to longer wavelength and also increases the absorption. Example: The effect of increasing conjugation on the absorption spectrum: 4 APPLICATION OF UV/VIS SPECTROSCOPY Detection of extent of conjugation The effect of increasing conjugation on the absorption spectrum when groups containing n electrons are conjugated with a π electron group; e.g; 5 APPLICATION OF UV/VIS SPECTROSCOPY Detection of extent of conjugation Aromatic systems, which contain p electrons, absorb strongly in the ultraviolet: 6 APPLICATION OF UV/VIS SPECTROSCOPY Detection of functional group: • UV spectroscopy is used to detect the presence or absence of chromophore in the compound. • The absence of a band in a particular band can be seen as an evidence for the absence of a particular functional groups. • If the spectrum of a compound comes out to be transparent above 200 nm than it confirms the absence of Conjugation, Carbonyl groups, Benzene or aromatic compounds and Bromo or Iodo atoms. 7 APPLICATION OF UV/VIS SPECTROSCOPY Detection of functional group: • Some of the most important chromophore (functional) groups are: 8 APPLICATION OF UV/VIS SPECTROSCOPY Detection of functional group: Example The spectrum of functional group benzene λmax 254 and Tolune λmax 261 9 APPLICATION OF UV/VIS SPECTROSCOPY Determination of the purity of substance: • Purity of a substance can also be determined with the help of UV spectroscopy. • The absorption of the sample solution is compared with the absorption of the reference solution. The intensity of the absorption can be used for the relative calculation of the purity of the sample substance. • UV absorption spectroscopy is one of the best methods for determination of impurities in organic molecules. • Additional peaks can be observed due to impurities in the sample and it can be compared with that of standard raw material. By also measuring the absorbance at specific wavelength, the impurities can be detected. Example: Benzene appears as a common impurity in cyclohexane. Its presence can be easily detected by its absorption at 255 nm. 10 APPLICATION OF UV/VIS SPECTROSCOPY Determination of the purity of substance: The impurities in cyclohexane is also detected by UV spectroscopy. 11 APPLICATION OF UV/VIS SPECTROSCOPY Identification of unknown compounds • An unknown compound can be identified with the help of UV spectroscopy. • The spectrum of unknown compound is compared with the spectrum of a reference compound and if both the spectrums coincide then it confirms the identification of the unknown substance. • UV spectroscopy is useful in the structure elucidation of organic molecules, the presence or absence of unsaturation, the presence of hetero atoms etc. From the location of peaks and combination of peaks, it can be concluded that whether the compound is saturated or unsaturated, hetero atoms are present or not etc. • There are many examples where the structure of an organic compound can be determined only by UV spectroscopy. 12 APPLICATION OF UV/VIS SPECTROSCOPY Identification of unknown compounds For example: • The following two isomeric compounds can be distinguished on the basis of their UV spectra. Compound I contain a conjugated diene system, which shows an absorption maximum at 232 nm; the calculated value is also 232 nm. Compound II absorbs near 175 nrn, the value for a single double bond, because its two double bonds are not conjugated. 13 APPLICATION OF UV/VIS SPECTROSCOPY Determination of configuration of geometric isomers: • It is observed that cis-alkenes absorb at different wavelength than the trans-alkenes. • The two isomers can be distinguished with each other when one of the isomers has non-coplanar structure due to steric hindrances. • The cis-isomer suffers distortion and absorbs at lower wavelength as compared to trans-isomer. • If the steric effect hinders the coplanarity of a system, delocalization of pi electrons will be hindered; and as a consequence, the length of conjugation will decrease. 14 APPLICATION OF UV/VIS SPECTROSCOPY Determination of configuration of geometric isomers: For example: As trans-cinnamic acid absorbs at a longer wavelength (λrnax = 272 nm, ԑrnax = 15,900) than cis-cinnamic acid (λrnax = 268 nm, ԑrnax = 10,700). This is because the former has a coplanar structure and delocalization of π electrons occurs completely across the molecule while in cis isomer the delocalization is hindered because it loses coplanarity owing to the overlap of ortho hydrogen of the phenyl group and the carboxylic group as shown in figure; 15 APPLICATION OF UV/VIS SPECTROSCOPY Quantitative Analysis: UV absorption spectroscopy can be used for the quantitative determination of compounds that absorb UV radiation. This determination is based on Beer’s law which is as follows: 𝑰𝟎 𝟏 𝑨 = 𝒍𝒐𝒈 = 𝒍𝒐𝒈 = − 𝒍𝒐𝒈 𝑻 = 𝒂𝒃𝒄 = ∈ 𝒃𝒄 𝑰𝒕 𝑻 Where “A” is absorbance of the sample, ε is extinction co-efficient (molar absorptivity), c is concentration, and b is the length of the cell that is used in UV spectrophotometer. Other methods for quantitative analysis are as follows. • calibration curve method • simultaneous multicomponent method • difference spectrophotometric method 16 • derivative spectrophotometric method APPLICATION OF UV/VIS SPECTROSCOPY Quantitative Analysis: For example: The molar absorptivity of methyl vinyl ketone dissolved in hexane is 14,000 M-1 cm-1 at 219 nm. The solvent in which the sample is dissolved is reported because molar absorptivity is not exactly the same in all solvents. Therefore, the UV spectrum of methyl vinyl ketone in hexane would be reported as λmax = 219 nm (ԑmax = 14,000, hexane). 17 APPLICATION OF UV/VIS SPECTROSCOPY Qualitative Analysis: • 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 spectra. • Qualitative analysis through spectrophotometric methods achieve fast and accurate result using only small sample quantity. • Qualitative analysis has proven highly useful in major forms of organic compounds and helps to ensure patient health and safety. 18 APPLICATION OF UV/VIS SPECTROSCOPY Qualitative Analysis: For example: UV absorption spectroscopy is generally used for characterizing aromatic compounds and aromatic olefins. One of the most common medication used around the word is paracetamol. Qualitative analysis assures that the measurement process of active ingredient is precise and free of impure compounds. The result from these methods provide the data that can be used for the quality control and consistency in the product formation. Having precise and accurate result assures that the proper dosage and measurements are used for the intended application. 19 APPLICATION OF UV/VIS SPECTROSCOPY Dissociation constants of acids and bases: • The pKa of a compound can be determined by UV spectroscopy if either the acidic form or the basic form of the compound absorbs UV radiation. 𝑨 𝒑𝑯 = 𝒑𝑲𝒂 + 𝒍𝒐𝒈 𝑯𝑨 𝐅𝐫𝐨𝐦 𝐭𝐡𝐞 𝐚𝐛𝐨𝐯𝐞 𝐞𝐪𝐮𝐚𝐭𝐢𝐨𝐧, 𝐭𝐡𝐞 pKa value can be calculated if the ratio of [A-] / [HA] is known at a particular pH and the ratio of [A-] / [HA] can be determined spectrophotometrically from the graph plotted between absorbance and wavelength at different pH values. 20 APPLICATION OF UV/VIS SPECTROSCOPY Dissociation constants of acids and bases: For example: The phenolate ion has a λmax at 287 nm. If the absorbance at 287 nm is monitored as a function of pH, the pKa of phenol can be ascertained by determining the pH at which exactly one-half the increase in absorbance has occurred. At this pH, half of the phenol has been converted into phenolate ion, so this pH is equal to the pKa of the compound. 21