Theory — UV-Visible Spectroscopy
1. The basic UV-Vis experiment
A UV-Vis spectrophotometer passes monochromatic light through a sample (typically dissolved in a transparent solvent and held in a 1 cm-path cuvette) and measures how much light passes through compared to a blank. The absorbance A is computed as A = log₁₀(I₀/I), where I₀ is the incident intensity and I is the transmitted intensity. Modern instruments scan over a range (e.g., 200-800 nm) to give the full absorption spectrum.
2. Electronic transitions
UV-Vis absorption corresponds to electronic transitions: a valence electron is promoted from a filled orbital to an empty (or partially-filled) higher-energy orbital. The energy gap determines the wavelength absorbed (E = hc/λ). Common transitions in organic molecules:
| Transition | Energy | Typical λ range | Examples |
|---|---|---|---|
| σ \u2192 σ* | Highest | 120-150 nm (vacuum UV) | Saturated alkanes; not useful in routine UV-Vis |
| n \u2192 σ* | High | 150-200 nm | Alcohols, ethers, halides; usually below detection range |
| π \u2192 π* | Variable (depends on conjugation) | 180-700 nm | Alkenes (170-180), conjugated dienes (220-260), aromatics (250-280), polyenes (300-700) |
| n \u2192 π* | Low (forbidden, weak) | 270-330 nm | Carbonyls (acetone n\u2192\u03c0* at 280 nm, ε~15) |
| d \u2192 d (transition metals) | Variable | 400-700 nm | Coloured transition metal complexes; KMnO\u2084 (525 nm), Cu²\u207A (810 nm) |
| Charge transfer | Variable | 200-700 nm | Metal complexes; donor-acceptor pairs; very intense (ε > 10,000) |
3. Chromophores and chromophore extension
A chromophore is a structural feature that absorbs UV-Vis light. Most chromophores in organic chemistry are based on π-bonded systems (alkenes, aromatics, carbonyls, azo, nitro) and the electrons promoted are typically in π or non-bonding (n) orbitals.
Conjugation extends and red-shifts λmax. When π-bonds are conjugated, the energy gap between the highest occupied (HOMO) and lowest unoccupied (LUMO) molecular orbitals decreases, so absorption moves to longer wavelengths. This is why β-carotene (11 conjugated C=C bonds) absorbs in the visible range (~450 nm, orange) while a single C=C absorbs in the deep UV (~170 nm, invisible to the eye). Conjugated polyenes are responsible for the colours of carotenoids, lycopene, retinal, and many natural and synthetic dyes.
| System | λmax (nm) | Notes |
|---|---|---|
| Single C=C (ethylene) | ~170 | Vacuum UV; not seen in routine spectra |
| 1,3-Butadiene (2 conjugated C=C) | ~217 | UV; typical conjugated diene |
| 1,3,5-Hexatriene (3 conjugated C=C) | ~258 | UV; structured |
| β-Carotene (11 conjugated C=C) | ~450 | Visible; orange colour |
| Lycopene (11 conjugated) | ~470 | Visible; red colour |
| Benzene (aromatic, 3 conjugated π) | ~254 (with vibrational structure) | UV; structured |
| Naphthalene | ~275 | Two fused aromatic rings |
| Anthracene | ~360 | Three fused aromatic rings; near-visible blue |
4. The Beer-Lambert Law
Absorbance is linearly related to concentration and path length:
where:
A = absorbance (dimensionless; log₁₀ ratio)
ε = molar absorptivity (L mol⁻¹ cm⁻¹)
c = molar concentration (mol L⁻¹)
l = path length (cm; typically 1.0)
For accurate quantitation, A should be in the range 0.2-1.0 (linear region). Above 1.0, dilute the sample. Below 0.2, concentrate or use a longer path length.
Molar absorptivity ε is a property of the molecule + the wavelength, not of the concentration. It quantifies how strongly a chromophore absorbs at a given λ. Strong chromophores (allowed transitions, intense colours) have ε = 10,000 to 100,000+. Weak chromophores (forbidden transitions, like n\u2192\u03c0* of carbonyls) have ε = 10-100. Knowing ε lets you calculate concentration from a measured absorbance.
5. Common chromophores and their typical λmax + ε values
| Chromophore | λmax (nm) | ε (M⁻¹ cm⁻¹) | Transition |
|---|---|---|---|
| Saturated alkane (C-H) | < 150 | -- | σ\u2192σ* |
| Alcohol/ether (-OH, -OR) | ~180 | ~200 | n\u2192σ* |
| Single C=C alkene | ~170 | ~10,000 | π\u2192π* |
| Acetone (n\u2192π*) | ~280 | ~15 | n\u2192π* (carbonyl forbidden) |
| Acetone (π\u2192π*) | ~190 | ~1000 | π\u2192π* (carbonyl, allowed) |
| Acrolein (CH\u2082=CH-CHO) | ~330 + 215 | ~25 + 12,000 | n\u2192π* + π\u2192π* |
| Benzene | ~254 | ~200 | π\u2192π* (Forbidden, structured) |
| Aniline (Ph-NH\u2082) | ~280 | ~1500 | π\u2192π* with NH\u2082 lone pair |
| Methyl orange (azo dye) | ~465 | ~25,000 | π\u2192π* extended; pH indicator |
| β-Carotene | ~450 | ~140,000 | π\u2192π* extended polyene |
| KMnO\u2084 (permanganate) | ~525 | ~2,500 | Charge transfer (LMCT) |
| Cu(H\u2082O)\u2086²\u207A | ~810 | ~10 | d-d (forbidden, weak) |
6. Solvent effects
Solvent affects λmax in two ways: (a) it must be transparent in the wavelength range of interest, (b) it can shift λmax via solvent-solute interactions. Solvent cutoff is the wavelength below which the solvent itself absorbs:
| Solvent | UV cutoff (nm) | Notes |
|---|---|---|
| Water | 190 | Best for very deep UV; excellent for charged or polar compounds |
| Methanol | 205 | Wide UV range; flammable; toxic |
| Ethanol | 205 | Similar to MeOH; less toxic |
| Acetonitrile | 190 | Best non-aqueous; good for all UV |
| Hexane / Cyclohexane | 200 | For non-polar compounds |
| Acetone | 330! | NOT useful below 330 nm; absorbs strongly itself |
| Diethyl ether | 220 | Limited; flammable |
| Chloroform | 245 | Limited UV range; halogen absorption |
Solvatochromism (solvent-induced shift): polar solvents red-shift π\u2192π* transitions (more polar excited state stabilised more by polar solvent) and blue-shift n\u2192π* transitions (lone pair H-bonded by polar solvent, lowering its energy and increasing the gap to π*).
7. Practical applications
- Concentration determination: If ε is known, A = εcl gives c. Routine for proteins (A₂₈₀ for tryptophan/tyrosine), DNA (A₂₆₀), drug compounds, dyes.
- Reaction monitoring: Watch a reactant disappear or product appear over time at a single λ.
- Purity check: A₂₆₀/A₂₈₀ ratio for nucleic acids; protein purity; impurity detection.
- Functional group/chromophore identification: Roughly identify aromatic, conjugated, carbonyl features.
- pH indicators: Methyl orange, phenolphthalein, etc. change λmax with pH \u2014 the colour change is the indicator.
- Tryptophan absorption (280 nm): standard for protein quantitation.
- Combined with chromatography (HPLC-UV): the most common HPLC detector.
Carbonyl n\u2192\u03c0*: ~280 nm, weak (ε ~15)
Carbonyl \u03c0\u2192\u03c0*: ~190 nm, strong (ε ~1000)
Benzene: ~254 nm, structured
Tryptophan / tyrosine: ~280 nm (protein quantitation)
DNA bases: ~260 nm
Conjugation extends: each new conjugated C=C adds ~30 nm to λmax
β-Carotene: ~450 nm (orange visible)
Methyl orange: ~465 nm (orange-red visible)
KMnO\u2084: ~525 nm (purple visible)
A λmax in the visible range (400-700 nm) means the compound is COLOURED. The colour you see is the COMPLEMENT of what is absorbed (e.g., absorbs ~450 nm blue light \u2192 you see orange-yellow).
8. Systematic UV-Vis analysis approach
- Identify the absorption maxima. Note the λmax values and approximate ε (from peak intensity).
- Match to chromophore type. λmax in UV (200-280)? Aromatic. λmax in near-UV (270-330)? Likely n\u2192\u03c0* (carbonyl). λmax in visible (400-700)? Conjugated or charge-transfer.
- For quantitation: Use Beer-Lambert at the chosen λ (use λmax for max sensitivity).
- For unknown structure: Combine with NMR (gives connectivity) and IR (gives functional groups). UV-Vis adds chromophore information.
Instructions
The Simulation has four parts. Complete in order.
Prerequisite: Familiarity with organic functional groups and the IR + Mass Spec + NMR labs. UV-Vis is the fourth and final lab in the spectroscopy quartet, completing the structural identification toolkit.
Simulation
Four interactive parts. Use the ↺ Reset Simulation button to clear all answers.
Eight chromophore-identification cases. For each: (a) wavelength region; (b) electronic transition; (c) chromophore type.
Six unknown cuvette samples. Click Acquire Spectrum, identify the compound from the UV-Vis spectrum. Peak labels appear after you answer.
Eight harder puzzles: Beer-Lambert quantitation, conjugation, solvent effects.
Round 1 — SDS interpretation
Four UV-Vis materials. Each has 4 questions.
Round 2 — Microscale sample prep
Six sample-prep scenarios.
Team Questions
Discuss with your team before answering.
Example Lab Notebook Entry
Use the format below as a template.
UV-Visible Spectroscopy — Lab Notebook Entry
Submitted by: [Student Name]
Course: Organic Chemistry I · Section: 201-A · Date: May 10, 2026
Objective
To learn the systematic interpretation of UV-Vis spectra: identifying the chromophore from λmax and ε; understanding how conjugation extends λmax; applying Beer-Lambert\'s law for quantitative concentration determination; recognising the limitations of UV-Vis (low specificity); and choosing appropriate solvents/cuvettes/lamps for accurate measurement.
Instrument bench results (Section II)
| Cuvette | Compound | λmax (nm) | ε (M⁻¹ cm⁻¹) | Visible colour |
|---|---|---|---|---|
| 1 | Methanol (control) | none above 205 | -- | colourless |
| 2 | Acetone | 280 (n\u2192π*) | ~15 | colourless |
| 3 | Benzene | 254 (\u03c0\u2192\u03c0*, structured) | ~200 | colourless |
| 4 | β-Carotene | 450 (\u03c0\u2192\u03c0*, extended polyene) | ~140,000 | orange-yellow |
| 5 | Methyl orange | 465 (azo \u03c0\u2192\u03c0*) | ~25,000 | orange-red |
| 6 | KMnO\u2084 (aqueous) | 525 (charge transfer) | ~2,500 | purple |
Discussion
UV-Vis spectroscopy probes the lowest-energy electronic transitions in a molecule. The basic principle: a photon of UV or visible light is absorbed when its energy exactly matches an electronic-transition gap (HOMO\u2192LUMO most often). The wavelength at which absorption is maximal (λmax) reflects the energy gap; the molar absorptivity (ε) reflects the transition probability (allowed vs forbidden, symmetric vs asymmetric).
For organic chemistry, the most useful transitions are π\u2192π* (typically ε = 10\u2074-10\u2075, intense) and n\u2192π* (typically ε = 10-100, weak because forbidden). Single C=C absorbs in the deep UV (~170 nm) and isn\'t seen in routine UV-Vis spectra; conjugated π-systems shift λmax progressively into the UV and visible. The classic series: 1,3-butadiene (217 nm) \u2192 1,3,5-hexatriene (258 nm) \u2192 longer polyenes (300+) \u2192 β-carotene\'s 11 conjugated C=C (450 nm). Each new conjugated C=C adds approximately 30 nm to λmax.
The instrument bench (Section II) demonstrates the entire range. Methanol shows no absorption above the solvent cutoff (~205 nm), serving as a clean baseline. Acetone shows a weak peak at 280 nm \u2014 this is the symmetry-forbidden n\u2192π* transition where a non-bonding lone pair on oxygen is promoted to the antibonding C=O π* orbital (low ε because the geometry change required for the transition is unfavourable). Benzene shows the structured \u03c0\u2192\u03c0* "B band" at 254 nm with vibrational fine structure (the multiple peaks come from coupling between the electronic transition and ring vibrations). Conjugated polyenes (β-carotene) show the extended \u03c0\u2192\u03c0* in the visible region. Azo dyes (methyl orange) show intense absorption from the electron-rich \u03c0-system around the N=N. Transition metal complexes (KMnO\u2084) show charge-transfer absorption (in this case ligand-to-metal: an electron from the O lone pair to the empty Mn 3d).
The Beer-Lambert law (A = εcl) is the basis for nearly all quantitative UV-Vis. For accurate quantitation, A should be in the linear range 0.2-1.0; outside this range, the response becomes non-linear (positive deviation from polychromatic light at high A; negative deviation from molecular interactions at very high concentration). Standard practice: prepare a calibration curve (5+ standards spanning the expected concentration range), measure A at λmax, plot A vs. c, fit a linear line, and use the slope (εl) to convert future A measurements to c.
Sample preparation matters. The cuvette must be transparent at the wavelength of interest \u2014 quartz (down to 200 nm) for UV; glass or polystyrene only for visible (above 320 nm). The solvent must not absorb at the analyte\'s λmax \u2014 acetone is unusable below 330 nm. Path length is typically 1.0 cm; for very dilute samples, longer (5-10 cm) cuvettes increase sensitivity. Concentration should be matched to give A in 0.2-1.0 range at λmax.
Compared to the other spectroscopic techniques in this quartet: UV-Vis tells us about chromophores and conjugation; IR identifies functional groups; NMR maps the carbon-hydrogen framework; MS determines molecular weight and fragmentation. UV-Vis is the LEAST specific (different compounds can have similar spectra), but it is FAST, INEXPENSIVE, and OFTEN USED FOR QUANTITATION (Beer-Lambert) where chromophore identity is already known.
Conclusion
UV-Vis spectroscopy is a workhorse analytical technique: simple, fast, sensitive, well-suited for quantitation. It complements the other spectroscopic methods (IR, NMR, MS) for full structural identification. Combined with Beer-Lambert quantitation, UV-Vis is widely used in pharmaceutical analysis, environmental monitoring, biochemistry, and chemistry teaching laboratories. The Spectroscopy quartet is now complete.
References
1. Pavia, D. L.; Lampman, G. M.; Kriz, G. S.; Vyvyan, J. R. Introduction to Spectroscopy, 5th ed., Cengage, 2015, Ch 2.
2. Silverstein, R. M.; Webster, F. X.; Kiemle, D. J. Spectrometric Identification of Organic Compounds, 8th ed., Wiley, 2014, Ch 7.
3. Skoog, D. A.; Holler, F. J.; Crouch, S. R. Principles of Instrumental Analysis, 7th ed., Cengage, 2017, Ch 13-14.
4. Perkampus, H.-H. UV-VIS Spectroscopy and Its Applications, Springer, 1992.
Practice Questions
Work through each before peeking at the hint.