ULTRAVIOLET/VISIBLE ABSORPTION SPECTROSCOPY

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1 ULTRAVIOLET/VISIBLE ABSORPTION SPECTROSCOPY Widely used in chemistry. Perhaps the most widely used in Biological Chemistry. Easy to do. Very easy to do wrong. MUJEEB KHAN BASIS SEMINAR 29th April 2009

2 OUTLINE INTRODUCTION ABSORPTION MECHANISM TERMINOLOGY ABSORPTION EFFECTING FACTORS INSTRUMENTATION APPLICATIONS

3 Introduction Electronic Excitation by UV/VIS Spectroscopy: X-ray: core electron excitation UV: valence electronic excitation IR: molecular vibrations Radio waves: Nuclear spin states (in a magnetic field)

4 Introduction Used to study molecules and their electronic transitions. Principle: The energy absorbed corresponds to the amount necessary to promote an electron from one orbital to another. Commonly used to determine the concentration of an absorbing species in solution (Quantitative Analysis) using Beer-Lambert law: 1,0 350 nm where: A = Absorbance I 0 = Intensity of the incident light I = Intensity of the light transmitted through the sample ε = Molar absorptivity (L mol -1 cm -1 ) l = sample path length (cm) c = Concentration of the solution (mol/l) ABSORBANCE 0,8 0,6 0,4 0,2 0, WAVELENGTH(nm)

5 UV/VIS Electronic Transitions Molecules have quantized energy levels. Bonding orbitals are lower in energy than antibonding orbitals. Non-bonding orbitals contains lone pair of electrons. As light absorbs electrons jumps from bonding or nonbonding orbital to the anti-bonding orbitals.

6 UV/VIS Electronic Transitions The Important Transitions are: from pi bonding orbitals to pi anti-bonding orbitals. from non-bonding orbitals to pi anti-bonding orbitals. from non-bonding orbitals to sigma anti-bonding orbitals. Groups in a molecule which absorb light are known as chromophores.

7 Absorption mechanism: A Case study of 1,3-Butadiene Has four π molecular orbitals Bonding orbitals are occupied Anti-bonding orbitals are unoccupied 1,3-Butadiene The interaction of the two double bonds with each other to produce a delocalised system of pi electrons over all four atoms is known as conjugation.

8 Absorption mechanism: A Case study of 1,3-Butadiene LUMO π* Energy hν (UV Irradiation) Four p atomic orbitals HOMO π Ground state electronic configuration Excited state electronic configuration

9 Terminology: Chromophore Chromophore: A covalently unsaturated group responsible for electronic absorption. or Any group of atoms that absorbs light whether or not a color is thereby produced. e.g. C=C, C=O, NO 2 etc. A compound containing chromophore is called chromogen. There are two types of chromophore: I. Independent chromophore: single chromophore is sufficient to import color to the compound e.g. Azo group II. Dependent chromophore: When more than one chromophore is required to produce color. e.g. acetone having one ketone group is colorless where as diacetyl having two ketone group is yellow.

10 Terminology: Auxochrome Auxochrome: A saturated group with non-bonding electron when attached to chromophore alters both wavelengths as well as intensity of absorption. e.g. OH, NH 2, NHR etc. Bathochromic group: The group which deepens the color of chromophore is called bathochromic group. e.g. Primary, secondary and tertiary amino groups. Bathochromic shift: (Red shift) shift of lambda max (λ max ) to longer side or less energy is called bathochromic shift or read shift. This is due to substitution or solvent effect. Hypsochromic shift: (Blue shift) shift of lambda max (λ max ) to shorter side and higher energy is called hypsochromic or blue shift. e.g solvent effect. Hyperchromic effect: an increase in absorption intensity Hypochromic effect: a decrease in absorption intensity Hypsochromic Hyperchromic Hypochromic Bathochromic 200 nm 800

11 Solvent Effects Different compounds may have very different absorption maxima and absorbances. Intensely absorbing compounds must be examined in dilute solution, so that significant light energy is received by the detector, and this requires the use of completely transparent (non-absorbing) solvents. Typical solvents are water, ethanol, hexane and cyclohexane. Solvents having double or triple bonds, or heavy atoms (e.g. S, Br & I) are generally avoided. Because the absorbance of a sample will be proportional to its molar concentration in the sample cuvette, a corrected absorption value known as the molar absorptivity is used when comparing the spectra of different compounds.

12 Solvent Effects UV ABSORPTION SPECTRA of 1,2,4-Triazine

13 Instrumentation Spectrometric instruments have a common set of general features. often, one technique is distinguished from another by differences in these features. Some specific features for the UV/VIS Experiment. Light Sources: Deuterium lamp, W Filament (halogen lamp) and Xe arc lamp. Wavelength Selectors: Filters and Monochromators. Sample Container: Fused silica, quartz and glass. Detectors: Phototube, PMT, photodiode, photodiode array, and CCD array.

14 Instrumentation Beckman DU640 UV/Vis spectrophotometer.

15 Instrumentation Software

16 Source: Deuterium Lamp

17 Source: Tungsten Filament

18 Source: Xenon Lamp Tube filled with Xe (or sometime a mixture of Hg and Xe), invented in 1940, commercialized in 1961 by Osram. Pass a low voltage DC current to excite Xe. The broad spectral output closely resembles a natural day light, and is often used in projection system (e.g. 15 kw IMAX system)

19 Optical Filters Filters can absorb light with dye molecules incorporated into the glass or gel They can also pass or reject bands of light becouse of interference effects with multiple layers of materials. They can select a rather narrow region of light to allow through to a detector They are useful when a specific, known band of radiation needs to be monitored.

20 Monochromator A monochromator disperses the light in order to select a narrow bandwidth. Both gratings and prisms can be used for this dispersion. Numerous instrumental designs are available to account for various optical requirements

21 Applications UV/VIS spectroscopy is routinely used for the quantitative determination of analytes solutions in transition metal ions and highly conjugated organic compounds. Solutions of transition metal ions can be coloured (i.e., absorb visible light) because d electrons within the metal atoms can be excited from one electronic state to another. While charge transfer complexes also give rise to colors, the colors are often too intense to be used for quantitative measurement. The absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length. Thus, for a fixed path length, UV/VIS spectroscopy can be used to determine the concentration of the absorber in a solution. Linl for the video

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