UV-Vis spectroscopy measures how much ultraviolet and visible light a sample absorbs at different wavelengths. A beam of light passes through your sample, and the instrument records which wavelengths were absorbed and by how much. Since every molecule absorbs a unique pattern of wavelengths, this tells you what’s in the sample and how concentrated it is. The technique covers wavelengths from roughly 190 to 900 nanometers, spanning the ultraviolet and visible light regions.
The Core Principle: Molecules Absorbing Light
When light hits a molecule, its energy can bump electrons from a lower energy state to a higher one. This is an electronic transition, and it only happens when the incoming light carries exactly the right amount of energy. Different molecular structures require different energies, which correspond to different wavelengths of light. A molecule with carbon-carbon double bonds, for instance, absorbs at different wavelengths than one with oxygen lone pairs.
Most UV-Vis work with organic compounds relies on two main types of electron jumps. In one, electrons in double bonds get promoted to a higher-energy state. In the other, non-bonding electrons (typically lone pairs on oxygen or nitrogen) make the jump instead. These transitions happen to fall in the 200 to 700 nm range, which is exactly where UV-Vis instruments operate. Molecules need at least one unsaturated group (like a double bond or aromatic ring) to absorb in this region, which is why saturated hydrocarbons are essentially invisible to UV-Vis.
Beer-Lambert Law: Turning Light Into Numbers
The relationship between light absorption and concentration follows a simple equation known as the Beer-Lambert law: A = εbc. Here, A is absorbance (the quantity the instrument measures), ε (epsilon) is the molar absorptivity of the substance (a constant that reflects how strongly it absorbs at a given wavelength), b is the path length the light travels through the sample (usually 1 cm), and c is the concentration of the substance.
This equation is what makes UV-Vis so practical. If you know ε for your compound and you measure A, you can solve directly for concentration. Protein quantification, for example, uses this principle routinely. Proteins absorb at 280 nm, and if you know the extinction coefficient for your specific protein, a single absorbance reading gives you concentration. For a rough estimate when no coefficient is available, a default value works for most proteins. Antibodies tend to have higher extinction coefficients than average, so they need a different default.
The law does have limits. It assumes a perfectly monochromatic light source, relatively low concentrations, and a sample that doesn’t scatter light. At very high concentrations, molecules begin interacting with each other and the neat linear relationship between absorbance and concentration breaks down. Highly scattering samples (cloudy solutions, suspensions) also cause deviations because the instrument can’t distinguish between light that was absorbed and light that was scattered away from the detector.
Inside the Instrument
A UV-Vis spectrophotometer has four main components arranged in sequence: a light source, a monochromator, a sample holder, and a detector.
Most instruments use two light sources to cover the full wavelength range. A deuterium lamp produces UV light, working well from about 200 to 350 nm. A tungsten-halogen lamp handles the visible range and extends into the near-infrared. At around 260 nm, both lamps produce similar intensities, but below that the deuterium lamp is roughly 100 times stronger. Above 350 nm, the tungsten lamp dominates by a similar factor. The instrument switches between them automatically as it scans across wavelengths.
The monochromator isolates one narrow wavelength at a time. White light from the source enters through a slit, hits a diffraction grating (a precision-ruled surface that spreads light into its component wavelengths, like a prism), and only the selected wavelength exits through a second slit toward the sample. The grating rotates during a scan to sweep through the full wavelength range.
In a single-beam setup, the monochromatic light (intensity I₀) passes through the sample, and whatever isn’t absorbed reaches the detector (intensity I). The instrument calculates absorbance from the ratio of these two values. Double-beam instruments split the light and send half through a reference cell simultaneously, which corrects for any drift in the lamp or background absorption from the solvent.
Choosing the Right Cuvette
The sample holder, typically a small rectangular cuvette, matters more than most people expect. The cuvette material has to be transparent at the wavelengths you’re measuring, and different materials have very different cutoffs.
Quartz cuvettes transmit light from 190 to 900 nm, covering the full UV-Vis range. Glass cuvettes only transmit above about 360 nm, which means they block most UV light. If your experiment involves UV wavelengths below 360 nm, quartz is the only option that gives accurate results. For visible-only measurements above 400 nm, glass works fine and costs less. Disposable plastic cuvettes are available for routine visible-range work, but they absorb even more UV light than glass.
The standard path length is 1 cm, which is the b value in the Beer-Lambert equation. Shorter path length cuvettes exist for highly concentrated samples, and longer ones for very dilute solutions.
How Solvents Affect Your Results
The solvent you dissolve your sample in can shift absorption peaks in predictable ways. A more polar solvent stabilizes whichever electronic state (ground or excited) is more polar. For molecules where the ground state is more polar than the excited state, increasing solvent polarity widens the energy gap between the two states. This means the molecule needs higher-energy (shorter-wavelength) light to make the transition, shifting the absorption peak toward the blue end of the spectrum.
The reverse happens when the excited state is more polar. A more polar solvent stabilizes the excited state, narrowing the energy gap and shifting absorption toward longer, redder wavelengths. These shifts are called hypsochromic (blue) and bathochromic (red) shifts, and they’re important to keep in mind when comparing spectra measured in different solvents. Changing from hexane to water, for example, can shift a peak by 10 to 20 nm or more.
Common Applications
UV-Vis is one of the most widely used analytical techniques in chemistry and biology, largely because it’s fast, inexpensive, and nondestructive.
- Protein concentration: Measuring absorbance at 280 nm is the standard method for quantifying proteins in solution. The technique can detect protein concentrations down to about 100 µg/ml. Bovine serum albumin (BSA), one of the most common protein standards, has a well-characterized extinction coefficient calibrated against a NIST reference standard.
- Reaction kinetics: Because UV-Vis readings are nearly instantaneous, you can track how fast a reaction proceeds by monitoring how absorbance at a specific wavelength changes over time. If a colored product forms or a colored reactant disappears, the absorbance curve maps directly to reaction progress.
- Purity assessment: The ratio of absorbance at two wavelengths reveals contamination. For nucleic acid samples, the 260/280 nm ratio indicates protein contamination, while the 260/230 nm ratio flags organic solvent or salt carryover.
- Color analysis: Any colored solution absorbs visible light, so UV-Vis characterizes dyes, pigments, food colorings, and water quality indicators. The wavelength of maximum absorbance corresponds to the complementary color of what you see.
What Affects Measurement Accuracy
Absorbance values between roughly 0.1 and 1.0 give the most reliable results. Below 0.1, the difference between I₀ and I is so small that detector noise becomes significant relative to the signal. Above about 2.0, so little light reaches the detector that readings become unreliable. If your sample reads too high, dilute it. If too low, use a longer path length cuvette or concentrate the sample.
Bubbles in the cuvette scatter light and produce artificially high absorbance readings. Fingerprints on the optical faces of the cuvette do the same. Always handle cuvettes by the frosted sides, and check for bubbles before measuring. Running a baseline scan with your solvent alone and subtracting it from the sample scan removes background absorption from the solvent, the cuvette walls, and any slight detector offset.