UV-Vis Spectrophotometry Basics

Study Notes

Spectrophotometry measures the absorbance of light by an analyte (substance) at a specific wavelength to determine concentration.
UV/VIS spectrophotometry uses ultraviolet and visible light.
Light excites electrons in atoms or molecules to higher energy levels, causing absorbance specific to each molecule.
When light passes through a substance, some light is absorbed, and the rest is transmitted.
Transmittance (T) is the ratio of the intensity of light exiting the sample to the intensity entering the sample.
Absorbance (A) is the negative logarithm of transmittance.
A = -log(T)

Absorbance is directly proportional to the absorptivity (a constant at each wavelength), path length (distance light travels through the sample), and concentration of the absorbing substance.
A = abc, where:
- a = absorptivity of the substance
- b = path length
- c = concentration of the substance
When concentration is in molarity, the Beer-Lambert law is written as A = εbc, where ε is the molar absorptivity coefficient.

σ → σ transition:* An electron from a bonding σ orbital is excited to the corresponding antibonding σ* orbital. High energy required; typically observed at shorter wavelengths (e.g., 125 nm). common in simple molecules like methane.
π → π transition:* An electron in a bonding π orbital is excited to the corresponding antibonding π* orbital. Occurs in molecules containing multiple bonds (alkenes, alkynes, etc.). Typical absorption region: 170-205 nm.
n → σ transition:* An electron from a nonbonding (n) orbital is excited to an antibonding σ* orbital. Requires less energy than σ → σ* transitions. Common in molecules containing atoms with lone pairs of electrons (e.g., oxygen, nitrogen). Absorption typically in the range of 150-250 nm.
n → π transition:* An electron from a nonbonding (n) orbital is excited to an antibonding π* orbital. Requires less energy and shows absorption at longer wavelengths (approximately 300 nm). Common in compounds with C=O, C=N, N=O groups.

σ → π*, and π → σ* transitions are theoretically possible but are usually observed with low intensity when they do occur.

The plot of absorbance (A) versus wavelength (λ) is known as an absorption spectrum.
Key features:
- λmax (wavelength at maximum absorbance)
- εmax (intensity of maximum absorbance)
Electronic transitions determine the absorption spectrum of a molecule

Chromophore: Part of a molecule responsible for color due to multiple bonds involved in absorption.
Non-conjugated alkenes: Show intense absorption below ~200 nm, making it difficult to measure.
Non-conjugated carbonyl groups: Show weak absorption in the 200-300 nm region.
Conjugation of C=C and carbonyl groups shifts λmax to longer wavelengths.

Auxochrome: Functional groups attached to a chromophore altering the wavelength or intensity of absorption. They do not have chromophoric properties on their own but affect those of chromophores they are attached to.

Bathochromic shift (red shift): Wavelength of maximum absorption shifts towards longer wavelengths.
Hypsochromic shift (blue shift): Wavelength of maximum absorption shifts towards shorter wavelengths.

Solvent effects on absorption intensity can be hyperchromic (increased intensity) or hypochromic (decreased intensity).
Polar solvents typically cause red shifts (bathochromic) in π → π* absorption peaks and blue shifts (hypsochromic) in n → π* transitions compared to nonpolar solvents.
This is often attributed to stabilization of the excited state in polar solutions.