UV-Visible Spectroscopy

How UV-vis works

Visible light is a band of electromagnetic radiation from roughly 380 nm (violet) to 750 nm (deep red); the ultraviolet stretches below visible, from ~200 nm at the air-cutoff to ~380 nm. Photons in this range carry 1.5 to 6 eV of energy — enough to excite valence electrons from filled molecular orbitals into empty ones. When a sample absorbs a photon, an electron jumps from a bonding or non-bonding orbital (π, n) to an antibonding orbital (π*, σ*). The absorbed wavelength corresponds to the energy gap between those orbitals.

A spectrometer compares the light intensity reaching the detector through the sample (I) against the light reaching it through a blank reference (I₀). Two equivalent observables:

Transmittance:   T = I / I₀                  (range 0 – 1)
Absorbance:      A = log₁₀(I₀ / I) = −log₁₀(T)

Absorbance is the better quantity because it stacks linearly: an A of 1 means 90% absorbed, A of 2 means 99%, A of 3 means 99.9%. Plot A against wavelength and you have a UV-vis spectrum, with peaks at the resonance wavelengths of the molecule's electronic transitions.

The Beer-Lambert law

The cornerstone equation:

A = ε c l

where A is absorbance (dimensionless), ε is the molar absorptivity (M⁻¹ cm⁻¹, a property of the molecule at the chosen wavelength), c is concentration (M), and l is the path length the light travels through the sample (cm). Knowing any three lets you solve for the fourth — most often, you measure A and l, look up ε, and back out c.

Worked example. Single-stranded DNA at 260 nm has an average ε of 8,919 M⁻¹ cm⁻¹ per nucleotide. A 1 cm cuvette of unknown ssDNA reads A_260 = 0.42. Concentration in nucleotides is c = A / (ε l) = 0.42 / 8919 = 4.71 × 10⁻⁵ M = 47.1 µM nucleotides. Multiply by the average mass of a nucleotide (~330 Da) to get mass concentration: 47.1 × 10⁻⁶ × 330 = 15.5 µg/mL. The standard shortcut for double-stranded DNA: A_260 = 1.0 corresponds to 50 µg/mL; for ssDNA, 33 µg/mL; for RNA, 40 µg/mL. NanoDrop instruments use these constants directly.

The DNA 260/280 ratio in the lab

Every molecular biology lab quantifies extracted DNA with a 1 µL drop on a NanoDrop. Two ratios decide whether the prep is usable:

Ratio	Pure DNA	Pure RNA	Pure protein	What a low value means
A_260 / A_280	1.8	2.0	~ 0.6	Protein contamination, too much A_280
A_260 / A_230	2.0 – 2.2	2.0 – 2.2	—	Phenol, guanidine, or salt carry-over

The diagnostic logic: nucleobases absorb at 260 nm with ε per nucleotide around 8,000–15,000 M⁻¹ cm⁻¹ (purines higher, pyrimidines lower). Aromatic side-chains of tryptophan and tyrosine give proteins a peak at 280 nm. The 230 nm shoulder picks up phenols and EDTA-buffer remnants. A "good" DNA prep — clean enough for restriction digestion, ligation, sequencing — meets both ratio targets and has A_260 above 0.1.

Common chromophores and their absorption

Chromophore	λ_max (nm)	ε (M⁻¹ cm⁻¹)	Transition	Where you see it
Isolated C=C (alkene)	~ 175	~ 10,000	π → π*	Below air-cutoff; not measurable
Conjugated diene	217	~ 21,000	π → π*	Butadiene; carotenoid analogues
C=O (ketone)	270 (weak), 188 (strong)	~ 15, ~ 1,100	n → π*, π → π*	Acetone, ester, aldehyde
Benzene	184, 204, 256	60,000, 7,900, 200	π → π*	Aromatics in pharma, dyes
DNA / nucleobases	260	8,919 (per nt avg)	π → π*	Quantitation standard
NADH	340	6,220	π → π*	Enzyme assays — coupled dehydrogenases
Tryptophan	280	5,500	π → π*	Protein quantitation
Tyrosine	274	1,400	π → π*	Protein A_280 contributor
Indigo (dye)	602	22,000	π → π*	Denim blue
β-carotene	450	140,000	π → π* (extended)	Carrots, food colour
Cu(H₂O)₆²⁺	800	10	d–d	Copper sulfate solution
MnO₄⁻	525	2,400	charge-transfer	Permanganate titrations

Two practical patterns. Conjugation length sets λ_max: each additional double bond adds ~30–40 nm (Woodward-Fieser rules formalise this). Allowed transitions (π → π* in flat aromatics) have ε of 10⁴ – 10⁵, while symmetry-forbidden d–d transitions in aqua-metal complexes have ε ≈ 1–100, which is why dilute Cu²⁺ looks pale blue while millimolar permanganate looks intensely purple.

UV-vis vs fluorescence vs phosphorescence

Technique	What it measures	Sensitivity	Selectivity	Best for
UV-vis absorption	Light absorbed (A vs λ)	~ 20 nM (strong chromophore)	Low — many things absorb	Routine concentration assay
Fluorescence emission	Light emitted after excitation	~ 1 pM (good fluorophore)	High — pick excitation + emission λ	Trace detection, imaging, qPCR
Phosphorescence	Slow emission from triplet state	Comparable to fluorescence	Very high — long lifetimes filter background	Lanthanide assays, time-resolved FRET
Circular dichroism (CD)	Differential absorption of L vs R circular light	Moderate	Stereochemistry-specific	Protein secondary structure, drug chirality
Diffuse reflectance UV-vis (DRS)	Reflected light from solid powders	Quantitative for solids	Like UV-vis	Heterogeneous catalysts, mineral analysis

Fluorescence beats absorption on sensitivity by a factor of ~10⁴ because a fluorophore radiates against a dark background, whereas an absorbing molecule has to dim a bright lamp by a measurable fraction. Use absorption when concentrations are µM to mM; use fluorescence below µM; use phosphorescence (or time-resolved fluorescence) when autofluorescence backgrounds drown the signal.

Hardware

• Light source. Deuterium arc lamp for 190–400 nm, tungsten halogen for 350–800 nm. The two switch over automatically around 350 nm.
• Monochromator. A diffraction grating + slits select a narrow band (typical 1–4 nm bandpass) to send through the sample. Bandwidth narrower than the absorption band keeps Beer-Lambert linear; wider bandwidth distorts peak shape.
• Sample compartment. Holds 1, 4 or 8 cuvettes. Temperature-controlled holders with stirrers handle kinetics. NanoDrop and 96-well plate readers replace cuvettes with droplet pedestals or microplate wells.
• Detector. Photomultiplier tube for low-light/wide range, photodiode or photodiode-array for fast multi-wavelength acquisition. Diode arrays capture 200–800 nm in 100 ms — essential for HPLC detectors.
• Cuvettes. Quartz for UV; glass for visible-only; plastic disposables for high-throughput. Wash with ethanol then water; never with acetone (etches polystyrene).

Real-world applications

• DNA quantification. Every molecular biology lab quantifies extracted DNA on a NanoDrop using A_260 with the 50 µg/mL × A_260 conversion for double-stranded DNA. The 260/280 ratio (target 1.8) and 260/230 ratio (target ≥ 2.0) flag protein and reagent contamination respectively.
• Enzyme kinetics. Coupled-enzyme assays watch NADH at 340 nm — dehydrogenase reactions either consume or produce NADH, and the rate of A_340 change times the cuvette dimensions gives turnover number. The pyruvate-kinase / lactate-dehydrogenase system tracks ATP-consuming kinases this way.
• Protein quantification. A_280 with ProtParam-derived ε (calculated from Trp, Tyr and disulfide counts) gives concentration to 5%. Bradford and Lowry assays use dye binding for less spec-friendly samples and read out at 595 or 750 nm.
• Drinking water testing. EPA-approved colorimetric methods quantify nitrate (220 nm direct, or 543 nm after Griess reaction), iron (510 nm with phenanthroline), chlorine (515 nm with DPD), and dozens more. Field-deployable handheld units sell for under $1,000.
• HPLC detection. A diode-array UV detector at 254 or 280 nm sits at the outlet of every analytical HPLC column. Peak area integrates to mass; full-spectrum acquisition lets a software match each chromatographic peak against a UV library.
• Pharmaceutical dissolution testing. USP requires UV monitoring of drug release from tablets into simulated gastric fluid; an automated robot pulls aliquots at 5, 15, 30, 60 minutes and reads them on a 96-cell spectrophotometer.
• Industrial colour control. Paint, ink, food and textile factories run quality-control UV-vis on every batch — a colour delta of more than three "tristimulus" units rejects the lot.

Common mistakes and pitfalls

• Reading absorbance above 1.5. Stray light makes A flatten; concentrations come out under-reported. Dilute the sample to land in 0.1 – 1.0 absorbance and re-measure.
• Forgetting to blank. Solvent, buffer salts, even cuvette glass absorb a little. Always blank against the matrix you're measuring in, not pure water.
• Using a plastic cuvette in the UV. Polystyrene cuts off below 340 nm; PMMA below 280 nm. Reading A_260 in PS gives nonsense — always quartz for UV.
• Ignoring scattering from particulates. Cloudy samples scatter light, mimicking absorbance with a wavelength-dependent baseline (~1/λ⁴). Filter through 0.22 µm or centrifuge before reading; baseline-subtract a smooth Rayleigh fit if you must.
• Trusting concentration above the linearity limit. Beer-Lambert holds when molecules don't interact. Aggregating dyes (cyanines, porphyrins) deviate sharply at concentrations above ~10 µM.
• Not warming the lamp. Deuterium and tungsten lamps drift in the first 15 minutes after switch-on. Quantitative work needs a warm-up period and a fresh blank.
• Confusing ε at one wavelength with another. Molar absorptivity is wavelength-specific. Quoting "ε = 8919" for DNA without specifying 260 nm will get you 50% errors at neighbouring wavelengths.
• Mixing up 260/280 and 280/260. The convention is the larger over the smaller, so DNA's 260/280 > 1. Some software flips it; check the axis label.