Molecular Spectroscopy (UV-Vis, IR, Fluorescence)
Molecular Spectroscopy (UV-Vis, IR, Fluorescence)
This domain tests how molecules interact with ultraviolet, visible, infrared and fluorescence radiation, and how spectra become quantitative or structural information.
Core rules
- Beer–Lambert: A = εbc. Absorbance is additive; use a reagent blank and keep A in the linear range, typically 0.1–1.0. Stray light and polychromatic radiation cause negative deviation at high absorbance.
- UV-Vis chromophores include conjugated systems, carbonyls and aromatic rings. The solvent must be transparent at the measurement wavelength.
- IR absorption requires a change in dipole moment. Characteristic frequencies include O–H 3200–3600 cm−1, C=O 1700–1750 cm−1 and C≡N 2220–2260 cm−1. FTIR uses an interferometer; ATR avoids KBr pellet water interference.
- Fluorescence occurs at longer wavelength than excitation. Quantum yield, quenching, inner-filter effects and photobleaching limit linearity. Fluorescence is more sensitive but less robust than absorbance.
Worked example
A solution in a 1.00 cm cell has A = 0.450 at 520 nm and ε = 1.25 × 10^4 L mol−1 cm−1. c = A/(εb) = 0.450/(1.25 × 10^4 × 1.00) = 3.60 × 10^-5 mol/L in the cuvette. If 5.00 mL was diluted to 100.0 mL, original c = 3.60 × 10^-5 × (100.0/5.00) = 7.20 × 10^-4 mol/L. The dilution factor is 20, not 0.05.
Traps
- Measuring at A > 1.5 and assuming linearity.
- Forgetting the blank or using a cuvette with a different path length.
- Assigning IR bands without checking the fingerprint region.
- Ignoring solvent cut-off in UV.
- Reporting fluorescence intensity without quenching controls.