Four techniques,one structure.

The questions each technique answers

Mass spectrometry tells you how heavy the molecule is and, at high resolution, its formula. Infrared spectroscopy tells you which functional groups are present. NMR spectroscopy tells you how the atoms are connected. UV–Vis spectroscopy tells you about conjugation and, above all, how much of a substance is in a solution.

A good structure determination uses them in that order: formula first, then functional groups, then connectivity, and finally a check that every signal is explained.

Start with the formula: degree of unsaturation

Once you know the molecular formula, count rings plus π bonds with the degree of unsaturation: (2C + 2 + N − H − X) / 2, where X is the number of halogens. Oxygen does not change it.

For C8H8O the result is (16 + 2 − 8) / 2 = 5. A benzene ring accounts for 4 (one ring and three C=C), so one more double bond or ring is left. Acetophenone, a benzene ring with a C=O, fits; so do several isomers, which is why you need the other spectra.

Mass spectrometry

The mass spectrometer ionises molecules and sorts the ions by mass-to-charge ratio, m/z. The molecular ion gives the molecular mass: M⁺• in electron ionisation, or [M+H]⁺ and similar ions in soft methods such as electrospray. High-resolution instruments measure the mass to about four decimal places, which narrows the formula to a few candidates.

Isotope patterns are quick clues. A compound with one chlorine shows an M+2 peak about one third the height of M, because ³⁵Cl and ³⁷Cl occur roughly 3 : 1. One bromine gives M and M+2 of almost equal height. Fragments help too: a loss of 15 suggests a methyl group, a loss of 18 water.

Infrared spectroscopy

IR spectra plot absorption against wavenumber in cm⁻¹. A few regions do most of the work. A strong, sharp band near 1700 cm⁻¹ means C=O: around 1715 cm⁻¹ for a simple ketone, around 1735–1750 cm⁻¹ for an ester, lower when conjugated. A broad band around 3200–3550 cm⁻¹ means O–H of an alcohol; carboxylic acids show a very broad O–H from about 2500 to 3300 cm⁻¹. A sharp band near 2250 cm⁻¹ points to a nitrile.

Below about 1500 cm⁻¹ lies the fingerprint region, full of overlapping bands. It is hard to assign but very characteristic of the whole molecule, so it is used to match a sample against a reference spectrum.

NMR spectroscopy

A ¹H NMR spectrum gives four kinds of information. The number of signals tells you how many sets of equivalent protons there are. The chemical shift δ, in ppm, tells you their environment: alkyl protons near 0.9–1.5 ppm, protons next to C=O near 2.0–2.5 ppm, protons on carbon bearing oxygen near 3.3–4.5 ppm, aromatic protons near 6.5–8 ppm and aldehyde protons near 9.5–10 ppm.

Integration gives the ratio of protons in each signal. Coupling gives the neighbours: in simple first-order spectra, a signal next to n equivalent protons splits into n + 1 lines. Ethyl acetate shows the pattern clearly: a quartet near 4.1 ppm (OCH2, 2H, next to CH3), a singlet near 2.0 ppm (the acetyl CH3, 3H, no neighbours) and a triplet near 1.3 ppm (CH3, 3H, next to CH2).

Shifts are given in ppm so that they do not depend on the instrument. On a 400 MHz spectrometer 1 ppm is 400 Hz; on 600 MHz it is 600 Hz. Coupling constants J are given in Hz and stay the same on any field, which is why multiplets look narrower in ppm on stronger magnets.

UV–Vis and the Beer–Lambert law

UV–Vis spectra come from electronic transitions, mainly in conjugated systems, aromatic rings and metal complexes. They say less about structure but are excellent for quantification. The Beer–Lambert law, A = εlc, links absorbance A to the molar absorptivity ε, the path length l and the concentration c.

With ε = 15,000 L mol⁻¹ cm⁻¹, a 1.00 cm cuvette and a measured absorbance of 0.450, the concentration is 0.450 / (15,000 × 1.00) = 3.00 × 10⁻⁵ mol/L. In practice you build a calibration curve from standards and stay inside its linear range, roughly A = 0.1 to 1.

Putting it together

Use the formula and degree of unsaturation to limit the options. Use IR to confirm or rule out functional groups. Use ¹H and ¹³C NMR to assemble the fragments and connect them. Then go back and check that every peak in every spectrum is explained by your proposed structure. If one is not, the structure is not finished. Predicted spectra are a useful cross-check, but they are predictions, not measurements, and should be labelled as such.