CM1512-PAH-uv-vis

Ultraviolet Spectroscopy (UV)

  • Definition: Technique that relies on the absorbance of energy due to electronic transitions in molecules.

  • Chromophore: The essential part of a molecule that absorbs UV or visible light, commonly includes:

    • Conjugated double bonds

    • Aromatic rings

    • Double bonds conjugated to heteroatoms

  • Applications: Used for both qualitative and quantitative analysis, with emphasis on the latter, especially combined with High-Performance Liquid Chromatography (HPLC).

  • Terminology: Term "UV-VIS" often used when measuring both UV and visible parts of the spectrum.

Color and Electronic Transitions in Spectroscopy

  • Examples of Colorations:

    • Pale blue Cu2+ results from electronic transitions between ligand orbitals and metal ion orbitals.

    • [MnO4]− transitions between p orbitals in conjugated organic compounds (e.g., β-carotene, azo dyes).

Complementary Colors

  • Concept: Spectral bands absorbed and perceived are opposite each other on the color wheel, leading to specific visual phenomena.

Absorption vs. Reflection

  • Color Perception: A colored object appears to possess a certain color by reflecting light of that wavelength rather than absorbing it.

Detailed Absorption Characteristics

  • Chlorophyll: The green pigment of plants, absorbs light at:

    • 450 nm (blue) and 700 nm (red)

    • Reflects light between 500 – 600 nm, hence appears green.

UV/Vis Spectra of Indicators

  • Phenolphthalein: Shows different absorbance in acidic and alkaline conditions across a defined pH range.

  • Methyl Orange & Litmus: Similar graphical representations of absorbance demonstrate how pH affects their color transitions.

Chromophores in Biological Context

  • Definition: Molecule parts that absorb UV/VIS radiation, specific functional groups include:

    • C=O (carbonyl groups)

    • Presence of delocalized electrons in conjugated systems.

Quantitative Analysis by UV Spectroscopy

  • Key Principle: There is a linear relationship between concentration, path length, and absorbance, as described by Beer-Lambert Law:

    • Formula: A = ecl (where A = absorbance, e = molar absorptivity, c = concentration, l = path length)

  • Measurement Standards: Samples should be measured at wavelengths near their respective λmax (wavelength of maximum absorbance).

Important Chromophores and Their Absorbance Maximums

  • Notable Compounds:

    • Ethene: max = 170 nm, Emax = 10,000

    • 1,3-Butadiene: max = 217 nm, Emax = 21,000

    • β-Carotene: max = 451 nm, Emax = 139,500

Application of Beer-Lambert Law in Solutions

  • Practical Use: To determine the concentration of a solution using absorbance data. Calibration curves can be helpful in specifying unknown concentrations from known samples.

Monitoring Biological Processes Using UV Spectra

  • Biochemical Reactions: Using UV spectra of NAD+ and NADH to monitor biochemical reactions, particularly at 340 nm due to different chromophore properties.

Role of UV Radiation in Vision

  • Visual Mechanism: 11-cis-retinal isomerizes to all-trans-retinal upon photon absorption, triggering the vision process across various organisms.