UV-Visible Spectroscopy

UV-Vis spectroscopy measures electronic transitions - jumps of electrons from lower to higher energy orbitals. Only molecules with readily excitable electrons (pi systems, lone pairs) absorb in the UV-Vis range. The technique is extensively used to quantify concentrations of absorbing species in solution (Beer-Lambert law) and to characterize conjugated molecules.

What Absorbs UV-Visible Light

A molecule absorbs UV-Vis light when the photon energy matches the gap between an occupied molecular orbital and an unoccupied one. The most common transitions:

• pi → pi*: a pi electron jumps from a bonding pi orbital to an antibonding pi* orbital. Occurs in alkenes, aromatics, and conjugated systems.
• n → pi*: a lone pair electron jumps to an antibonding pi* orbital. Occurs in carbonyls and other molecules with both lone pairs and pi bonds.
• sigma → sigma*: very high energy, requires short UV (not usually measured in typical UV-Vis).

For organic chemistry, the most important transitions are the pi → pi* transitions in conjugated pi systems.

Conjugation and Absorption Wavelength

The more conjugated the pi system, the smaller the HOMO-LUMO gap, and the LONGER the wavelength of absorbed light:

This is why beta-carotene (in carrots, tomatoes) appears orange: it absorbs blue light (~450 nm) and the remaining reflected/transmitted light appears orange. Lycopene (11 conjugated C=C) appears red. Chlorophyll appears green.

The Rule: More Conjugation → Longer λmax

Each additional conjugated double bond extends the pi system. In molecular orbital terms, more p orbitals combining means the MOs spread out: the HOMO moves UP in energy and the LUMO moves DOWN. The gap shrinks. Lower-energy photons (longer wavelength) can bridge the gap.

Chromophores

A chromophore is any structural unit that absorbs UV-Vis light. Common organic chromophores:

• Isolated C=C: ~170-180 nm.
• Conjugated dienes: ~210-230 nm.
• Isolated C=O: ~290 nm (n → pi*, weak).
• Aromatic ring (benzene): 180, 200, 255 nm.
• Extended conjugation: 250-700+ nm.

Transition metal complexes also absorb in the visible region, giving them their color (Cu²⁺ = blue; Fe³⁺ in hemoglobin = red, etc.).

Solvatochromism

The exact λmax of a compound can shift based on the solvent because solvent-solute interactions stabilize different electronic states. Measuring this shift gives additional information about the electronic structure. Not usually tested on the MCAT but appears in research passages.

Quantification with Beer-Lambert

UV-Vis is most commonly used to quantify concentration using the Beer-Lambert law (Section 11.5):

A = εbc

where A is absorbance, ε is molar absorptivity, b is path length (usually 1 cm), and c is concentration. This linear relationship (for dilute solutions) allows unknown concentrations to be determined from measured absorbance.

Biochemistry Applications

• Protein concentration: measured at 280 nm (tryptophan and tyrosine absorb).
• DNA/RNA concentration: measured at 260 nm (bases absorb). A $\frac{260}{280}$ ratio > 1.8 indicates high purity nucleic acid; < 1.6 indicates protein contamination.
• Enzyme assays: NADH vs. NAD⁺ absorbance difference at 340 nm is used in countless enzyme kinetics experiments.
• Photosynthesis pigments: chlorophyll absorbs at ~430 and 665 nm.