UV-Vis spectroscopy is a technique that measures how much ultraviolet and visible light a sample absorbs at different wavelengths. It matters because absorption reveals information about a molecule's electronic structure, especially the presence of chromophores such as conjugated double bonds or metal complexes. The method is widely used in chemistry, biochemistry, environmental testing, and materials science.
A UV-Vis spectrum can identify absorbing species and measure concentration quickly and nondestructively in many cases.
Inside a spectrophotometer, light from a UV and visible source is separated by wavelength, passes through a cuvette containing the sample, and reaches a detector. When photons have the right energy, electrons in the sample can move to higher energy levels, causing absorbance at specific wavelengths. The resulting absorption spectrum plots absorbance versus wavelength and often shows peaks at wavelengths where electronic transitions are most likely.
Quantitative analysis usually uses the Beer-Lambert law, A = εbc, which connects absorbance to concentration, path length, and molar absorptivity.
Understanding Chemistry: UV-Vis Spectroscopy
Electronic transitions are not all equally likely. In many organic molecules, electrons in double bonds can be promoted from a bonding orbital to a higher energy antibonding orbital. Molecules with several linked double bonds have delocalised electrons spread across a larger region.
This usually reduces the energy gap between levels. They can then absorb longer wavelengths than molecules with only one isolated double bond. This pattern explains why compounds such as beta carotene absorb visible light, while many small organic molecules absorb only ultraviolet light.
In metal complexes, electrons may move between metal and ligand orbitals, or between different metal orbitals. These transitions often create intense colours.
The colour seen by the eye is usually not the colour absorbed most strongly. A solution that absorbs blue light may look orange, since orange is roughly the complementary visible colour. A red dye transmits or reflects more red light while removing other parts of the spectrum.
This is why a spectrum can help explain the appearance of food colourings, plant pigments, inks, stained glass, and transition metal solutions. Colour is affected by the chemical environment too. Changes in acidity can alter a molecule’s electron arrangement.
Indicators change colour because their protonated and deprotonated forms have different absorption patterns. Solvents can shift or broaden peaks by interacting with the absorbing molecules.
For reliable concentration measurements, students need to control several practical details. The blank sample should contain everything except the substance being measured, often the solvent and any reagents. It corrects for light absorbed by the cuvette, solvent, and instrument parts.
Cuvettes must be clean, matched where possible, and held in the same orientation. Fingerprints, scratches, bubbles, or cloudy particles scatter light and can give falsely high absorbance values.
Visible light can use glass or plastic cuvettes in many cases, but ultraviolet measurements commonly need quartz because ordinary glass absorbs much ultraviolet light. Measurements are often made near the wavelength of strongest absorption because the signal is clearer there.
The relationship between absorbance and concentration has limits. It works best for dilute, clear solutions containing one stable absorbing species. At high concentration, molecules can interact with each other and change how they absorb.
Stray light inside the instrument can make strong absorption appear weaker than it really is. Chemical reactions during a measurement can change the spectrum over time. A calibration graph made from standards is therefore more trustworthy than relying only on a theoretical value.
When reading a spectrum, pay attention to peak position, peak height, peak width, and the baseline. A single peak is rarely final proof of identity, but it becomes useful evidence when combined with chemical tests or another analytical method.
Key Facts
- Absorbance is defined as A = log10(I0 / I), where I0 is incident light intensity and I is transmitted light intensity.
- Beer-Lambert law: A = εbc, where ε is molar absorptivity, b is path length, and c is concentration.
- Photon energy is related to wavelength by E = hc / λ, so shorter wavelengths have higher energy.
- UV-Vis spectra commonly cover about 200 to 800 nm, including ultraviolet and visible light.
- The wavelength of maximum absorbance is called λmax and is useful for identification and calibration.
- A chromophore is a part of a molecule that absorbs UV or visible light due to electronic transitions.
Vocabulary
- Absorbance
- Absorbance is a logarithmic measure of how much light a sample absorbs at a given wavelength.
- Transmittance
- Transmittance is the fraction of incoming light that passes through a sample, often written as T = I / I0.
- Chromophore
- A chromophore is an atom or group of atoms in a molecule responsible for absorbing UV or visible light.
- λmax
- λmax is the wavelength at which a substance shows its highest absorbance in a spectrum.
- Molar absorptivity
- Molar absorptivity is a constant that describes how strongly a substance absorbs light at a specific wavelength.
Common Mistakes to Avoid
- Using percent transmittance as if it were absorbance is wrong because absorbance is logarithmic, not a simple percentage.
- Forgetting to blank the spectrophotometer is wrong because the solvent and cuvette can absorb or scatter light and must be subtracted from the sample measurement.
- Assuming every peak identifies a compound by itself is wrong because different substances can have overlapping absorption bands and similar λmax values.
- Applying Beer-Lambert law at very high concentration is risky because concentrated samples can deviate from linear behavior due to interactions, stray light, or instrument limits.
Practice Questions
- 1 A solution has I0 = 100 units and I = 25 units at 520 nm. Calculate the absorbance using A = log10(I0 / I).
- 2 A compound has ε = 15000 L mol^-1 cm^-1 at λmax, the cuvette path length is 1.00 cm, and the measured absorbance is 0.450. Calculate the concentration in mol/L.
- 3 Two solutions of the same dye are measured at the dye's λmax in identical cuvettes. One gives A = 0.20 and the other gives A = 0.80. Explain which solution is more concentrated and why the comparison is valid.