What is UV-Vis spectroscopy?

The physics of the measurement

In UV–Vis spectroscopy, light spanning the ultraviolet and visible range passes through a sample and the instrument measures how much is absorbed at each wavelength. Absorption occurs when a photon’s energy matches the gap between electronic energy levels in a molecule, promoting an electron to a higher state; this is why coloured compounds and molecules with conjugated or aromatic systems absorb strongly. Plotting absorbance against wavelength produces a spectrum whose peaks indicate the wavelengths a substance absorbs, helping to identify and characterise it. The technique is one branch of the broader field of spectroscopy.

The Beer-Lambert law

The quantitative power of UV–Vis comes from the Beer–Lambert law, which states that absorbance is directly proportional to the concentration of the absorbing substance and to the path length of light through the sample (A = εcl).

The constant ε, the molar absorptivity, is characteristic of the substance at a given wavelength. By measuring absorbance at a peak and comparing it with a calibration standard, the concentration of an unknown can be calculated. The law holds best for dilute solutions, where absorbance varies linearly with concentration.

Uses in research

UV–Vis spectroscopy is a fast, inexpensive method for quantifying coloured and ultraviolet-absorbing substances across chemistry, biochemistry, and environmental science. Typical uses include measuring nucleic-acid and protein concentrations, monitoring reaction progress, and determining the concentration of dyes or metal complexes. It is frequently the detection step in HPLC. Reliable quantification depends on a properly fitted calibration curve and a clean baseline, so that absorbance readings are accurate and comparable.