Chemistry · Analytical and physical chemistry · Year 2

Spectra: determining structure

What is an unknown white powder? UV, IR, NMR and mass spectra can tell

The situation

An unknown white powder has been brought to a forensic chemistry laboratory: is it a drug or something else? The same question arises at the Institute of the Chemistry of Plant Substances of the Academy of Sciences of Uzbekistan every time a new compound is isolated from a plant: what is its structure? A molecule cannot be seen with the eye. The chemist records its UV, IR, NMR and mass spectra: each spectrum speaks about one part of the molecule, and they must fit together like pieces of a jigsaw puzzle. How do we assemble a structure from the peaks in a spectrum?

Topic

Structure determination: UV, IR, NMR and mass spectra. Forensic chemistry laboratory and the Institute of the Chemistry of Plant Substances: an unknown white powder has arrived. Recording the UV, IR, NMR and mass spectra one by one, solving the structure like a puzzle…

Key ideas

Characteristic IR frequencies
NMR: shift and the n + 1 rule
Molecular ion and isotopes

Formulas

E = h ν = \frac{h c}{λ}, \tilde{ν} = \frac{1}{λ}

Photon energy: UV and visible light excite valence electrons, IR light excites vibrations, and radio waves excite nuclear spins in a magnetic field.

\tilde{ν} = \frac{1}{2 π c} \sqrt{\frac{k}{μ}}, μ = \frac{m_{1} m_{2}}{m_{1} + m_{2}}

Harmonic oscillator: the stiffer the bond ( $k$ ) and the lighter the atoms ( $μ$ ), the higher the stretching frequency.

δ = \frac{ν - ν_{TMS}}{ν_{0}} \cdot 10^{6} (ppm), n \to n + 1 lines

Chemical shift (relative to TMS), and $n$ equivalent neighbouring protons split a signal into $n + 1$ lines (intensities follow Pascal's triangle).

r = \frac{1}{B} \sqrt{\frac{2 m U}{z e}}

Radius of the path of an ion accelerated through a voltage $U$ in a magnetic analyser: ions separate according to $m / z$ .

Ω = C - \frac{H}{2} + \frac{N}{2} + 1

The degree of unsaturation Ω is the number of rings plus double bonds (halogens count like H); for example, 4 for $C_{6} H_{6}$ .

Theory

Methods of analysis and spectroscopy

Methods of analysis are divided into chemical (gravimetry, titrimetry — based on a reaction), physicochemical (electrochemical, optical, chromatographic) and physical (spectroscopy, NMR, mass spectrometry, X-ray) methods. In spectroscopy a substance absorbs or emits electromagnetic radiation: gamma rays — nuclear processes, X-rays — inner electrons and crystal structure, UV and visible — transitions of valence electrons, IR — vibrations of atoms, microwaves — rotation of molecules and EPR, radio waves — nuclear spins in a magnetic field (NMR). In the second half of the twentieth century these instruments almost displaced classical chemical analysis: from a few milligrams of a substance the structure can be determined in a few hours. Atomic absorption and atomic emission methods, the Beer–Lambert law and quantitative photometry are covered in the “Optical methods of analysis” experiment.

UV and visible spectra

Electronic transitions: $σ \to σ^{*}$ (< 150 nm), $n \to σ^{*}$ (alcohols, ≈ 180 nm), $π \to π^{*}$ (strong, $ε$ ≈ 10⁴) and $n \to π^{*}$ (weak, $ε$ < 100). Chromophores are C=C, C=O, N=N, NO₂; auxochromes are OH, NH₂, Cl: attached to a chromophore, they shift the absorption to longer wavelengths (bathochromic shift); a shift to shorter wavelengths is hypsochromic. As conjugation gets longer, the bathochromic shift grows: ethene 171 nm, buta-1,3-diene 217 nm, hexa-1,3,5-triene 258 nm, β-carotene (11 C=C) ≈ 450 nm — it absorbs blue light and looks orange; acetylene derivatives obey the same rule. When phenol turns into phenolate in alkali, the band shifts to longer wavelengths; when aniline is protonated in acid (the NH₃⁺ group has no free electron pair left), it shifts to shorter wavelengths. The $n \to π^{*}$ band of acetone is at 279 nm in hexane and 264.5 nm in water: hydrogen bonding stabilises the n electrons. Franck–Condon principle: an electronic transition (10⁻¹⁵ s) is much faster than the motion of the nuclei, so the transition is “vertical” between the potential curves — which is why the band has vibrational structure. An excited molecule can release its energy as fluorescence (S₁ → S₀, nanoseconds) or phosphorescence (T₁ → S₀, spin-forbidden — milliseconds to seconds). Raman scattering: the frequency of the scattered light is shifted by the vibrational frequency; symmetric vibrations that are invisible in IR show up in Raman. Sample: a dilute solution ( $A$ ≈ 0.2–1), a quartz cuvette for wavelengths shorter than 320 nm; air absorbs light shorter than 190 nm (vacuum UV). The Woodward–Fieser rules and quantum-chemistry programs make it possible to calculate $λ_{\max}$ and compare it with experiment.

IR spectroscopy

A vibration absorbs IR light only if it changes the dipole moment. Stretching vibrations change bond lengths, bending vibrations change angles; the CH₃, CH₂ and NH₂ groups have symmetric and asymmetric versions (aniline N–H: 3433 and 3356 cm⁻¹). For a harmonic oscillator the only allowed transition is $Δ v = \pm 1$ ; a real bond is anharmonic (Morse potential), so weak overtones also appear — a little below 2ν (acetone C=O: 1715 → ≈ 3420 cm⁻¹). Characteristic frequencies: O–H free 3600–3650, hydrogen-bonded 3200–3550 (broad), carboxylic acids 2500–3300 (very broad); N–H 3300–3500; C–H sp³ 2850–2960, sp² 3000–3100, aldehyde 2720 and 2820; C≡N ≈ 2250; C=O: ketone ≈ 1715, ester ≈ 1735–1750, aldehyde ≈ 1725 (aromatic ≈ 1700), acid ≈ 1710, amide 1650–1690. Conjugation lowers the C=O frequency by 20–30 cm⁻¹, ring strain raises it (cyclopentanone ≈ 1745). Below 1500 cm⁻¹ lies the “fingerprint” region, unique to each substance. Hydrogen bonds: in a pure alcohol the O–H band is broad and at a lower frequency; on dilution in CCl₄ the intermolecular bonds break and a narrow “free” O–H band (3630) appears; an intramolecular bond, however, survives dilution. In saturated cyclic alcohols (cyclohexanol) the C–O vibrations of conformers with equatorial and axial OH absorb at different frequencies. Sample: a KBr disc, an ATR crystal, or a liquid film between NaCl plates (water dissolves the plates!). Computer programs (HyperChem, ChemOffice) calculate the normal vibrations; a harmonic calculation usually comes out a few per cent too high and is corrected with a scaling factor.

NMR and EPR

Nuclei with spin $I = \frac{1}{2}$ (¹H, ¹³C, ¹⁹F, ³¹P) split into two energy states in a magnetic field; the resonance frequency is $ν = γ B_{0} / 2 π$ (for ¹H 42.58 MHz/T: 400 MHz at 9.4 T). Electrons “shield” the nucleus from the field (shielding): a proton next to an electronegative atom is shielded less and appears at a larger $δ$ . Typical shifts: C–CH₃ 0.9–1.3; CH₃–C=O ≈ 2.1; O–CH₂ 3.4–4.2; alkene 4.5–6.5; aromatic 6.5–8.2; aldehyde 9.5–10; carboxylic acid 10–13 ppm. The integral is proportional to the number of protons. Spin–spin coupling: $n$ equivalent neighbouring protons split a signal into $n + 1$ lines; equivalent protons do not split one another. Geminal (on the same carbon, ²J) and vicinal (on adjacent carbons, ³J) coupling constants; with free rotation ³J is usually 6–8 Hz, but it depends on the angle between the two C–H bonds (Karplus): in trans-alkenes 12–18, in cis 6–12 Hz, in cyclohexane axial–axial 8–13, axial–equatorial 2–5 Hz — this is how the three-dimensional structure of a molecule is determined. $J$ in Hz does not depend on the field, so in a strong magnet multiplets become narrower on the ppm scale. OH and NH protons exchange — a broad signal that disappears when D₂O is added. ¹³C NMR: natural abundance 1.1 %, shifts 0–220 ppm; when coupling to protons is suppressed, each carbon gives a single line; in the “off-resonance” method CH₃ is a quartet, CH₂ a triplet, CH a doublet, C a singlet. Sample: 5–20 mg of substance in 0.6 mL of deuterated solvent (CDCl₃, DMSO-d₆), TMS as the standard. ChemOffice predicts shifts from increments. EPR is the resonance of an unpaired electron (radicals, Cu²⁺ complexes): $g$ ≈ 2.0023, in the X band ≈ 9.5 GHz and ≈ 0.34 T; $n$ equivalent protons split the hyperfine structure into $n + 1$ lines (CH₃• — 1 : 3 : 3 : 1). The physics is in the “Spin, the Zeeman effect and MRI” experiment.

Mass spectrometry

Ionisation: electron impact (EI, 70 eV — molecular ion M⁺• and many fragments), chemical ionisation (soft, [M + H]⁺), electrospray (ESI — multiply charged ions of proteins [M + nH]ⁿ⁺, $m / z$ = (M + n)/n), MALDI. Analysers: magnetic sector ( $r \propto \sqrt{m / z}$ ), quadrupole, time of flight; detectors: the Faraday cup (collects the charge and measures isotope ratios accurately) and the electron multiplier (a chain of dynodes that amplifies the signal a million times). A spectrum is intensity against $m / z$ , with the tallest peak set to 100 %. M⁺• gives the molecular mass; for inorganic substances the elemental and isotopic composition is determined. Isotope patterns: each C atom adds ≈ 1.1 % to M + 1; Cl — M : (M + 2) ≈ 3 : 1, Br — ≈ 1 : 1. Nitrogen rule: an odd M means an odd number of nitrogen atoms. Fragmentation: α-cleavage (alcohols → CH₂=OH⁺, $m / z$ 31; ketones → acylium CH₃CO⁺, $m / z$ 43), phenyl C₆H₅⁺ ( $m / z$ 77), tropylium ( $m / z$ 91); rearrangement ions — the McLafferty rearrangement (a γ-hydrogen migrates and the β-bond breaks: hexan-2-one → $m / z$ 58); metastable ions decompose before they reach the analyser and give a broad, diffuse peak at the fractional value $m^{*} = m_{2}^{2} / m_{1}$ . The physics of ion separation is in the “Charged-particle accelerator” experiment; combined with a gas chromatograph (GC–MS), in the “Extraction and chromatography” experiment.

An unknown substance, the laboratory and safety

Strategy: from MS the molecular mass, isotopes and nitrogen rule → formula; degree of unsaturation (rings + double bonds); IR — functional groups (C=O, O–H, N–H, C≡N); ¹H NMR — types, numbers and neighbours of protons; ¹³C — number and type of carbons; UV — conjugation. All the data must fit a single structure. In complex compounds IR shows the shift of a ligand on coordination (for example C≡N, C=O), UV–visible shows d–d transitions, and EPR shows Cu²⁺ (the “Complex compounds and ligands” experiment). Safety: the field of an NMR magnet is always on — people with pacemakers must not approach, and iron tools are pulled towards the magnet; the magnet is cooled with liquid helium and nitrogen — there is a risk of cold burns and of oxygen depletion in the room; do not look at a UV lamp; a mass spectrometer uses high voltage and vacuum; CDCl₃ and CCl₄ are handled in a fume cupboard. Model: the spectra are synthesised from peak positions in the literature (band widths, IR depths and fragment intensities are approximate); NMR uses the first-order ( $n + 1$ ) approximation, and aromatic multiplets are simplified; isotope patterns are calculated exactly from IUPAC abundances; the vibration amplitude is magnified ≈ 15 times and slowed down a million million times; the structure of the model X is revealed after the correct answer.

Did you know?

Felix Bloch and Edward Purcell received the 1952 Nobel Prize in Physics for the discovery of nuclear magnetic resonance; MRI scanners in hospitals are based on the same phenomenon.
Francis Aston was awarded the 1922 Nobel Prize in Chemistry for discovering the isotopes of many elements with the mass spectrograph he built.

Inside the experiment

The experiment starts from a real-life situation: you predict the result, change the controls, run the experiment and draw a conclusion. Inside are missions, theory, a quiz and a guided tour in which the camera moves and explains the key points.

More experiments in this section

Electrochemical analysis — Year 1
Qualitative analysis and gravimetry — Year 2
Titrimetric analysis — Year 2
Optical methods of analysis — Year 2
Extraction and chromatography — Year 2
Alloys: phase diagrams — Year 2
Chemical kinetics and Arrhenius — Year 2
Catalysis and enzymes — Year 2
Colloids and adsorption — Year 2
Computational chemistry and docking — Year 2