Soft ionisation and hard ionisation

Ionisation methods are commonly classified into ‘soft’ and ‘hard’ categories, with the distinction based on the amount of internal energy transferred to the sample molecules during the ionisation process. If the energy transferred is low and the molecules remain intact after ionisation, this is known as soft ionisation; if the energy transferred is high and the molecules break apart extensively during ionisation, this is known as hard ionisation. This single character difference determines the appearance of the spectrum and the types of samples for which the method is suitable.

Soft ionisation involves the transfer of a small amount of energy; molecules are less likely to fragment during ionisation, primarily yielding molecular ions and adduct ions, with very few fragments. Electrospray ionisation (ESI), matrix-assisted laser desorption/ionisation (MALDI), desorption electrospray ionisation (DESI) and atmospheric pressure photochemical ionisation (APPI/PI) all fall under the category of soft ionisation. Its advantages include the ability to directly determine molecular weight and its suitability for thermally unstable molecules and macromolecules; the drawback is that there are few fragments and structural information is relatively limited; for detailed structural identification, tandem mass spectrometry is often required to supplement the fragments.

Hard ionisation transfers more energy, causing extensive fragmentation of the molecular ion and generating a wealth of fragments. Electron ionisation (EI) is a typical example; its spectra are highly characteristic and can be compared against standard spectral libraries, making it highly suitable for the qualitative analysis of known small molecules; however, the molecular ion is often very weak or even absent, and the method is not well-suited to thermally unstable compounds—the molecules are fragmented before they can be analysed.

In mass spectrometry imaging, soft ionisation is almost invariably the preferred choice. Lipids, metabolites and drugs in biological tissues are mostly thermally unstable, and our focus is on the spatial distribution of their ‘intact molecules’; the intense fragmentation caused by hard ionisation not only destroys the molecules but also renders the spectra difficult to trace back to their origin. Neo Source INSTRUMENT’s MSI DPI and MSI LDPI both employ soft ionisation approaches: Through photochemical single-photon ionisation, neutral molecules are gently ‘illuminated’ to yield clean spectra dominated by molecular ions. Combined with high-resolution dual-field reflection TOF, this enables the acquisition of reliable molecular distributions without damaging the sample—even after imaging, the sections can still undergo H&E histological staining. Both sources can be fitted to mass spectrometers from Agilent, AB SCIEX and Thermo.