Laser-induced fluorescence spectroscopy was carried out on microcrystalline samples of three typical matrices under conditions of matrix-assisted laser desorption/ionization (MALDI). The emitted fluorescence intensity was determined as a function of incident laser fluence and a sublinear increase of the fluorescence intensity with laser fluence was found. A very good fit was obtained when the experimental fluorescence vs. fluence data were compared with a numerical model assuming that under typical MALDI fluence conditions a large fraction of molecules in the excited singlet state undergoes singlet-singlet annihilation. Throughout the fluence range relevant for MALDI, however, the experimental data could not be fit well to a model assuming resonant two-photon absorption as the process depopulating the singlet state. In a separate set of experiments, the singlet lifetimes of several typical crystalline MALDI matrices were determined and found to be considerably shorter than previously reported. While both singlet-singlet annihilation and resonant two-photon absorption have been discussed in the literature as candidates for pathways to primary matrix ion generation in MALDI, the data presented here suggest that singlet-singlet annihilation is the dominant mechanism for depopulating the singlet state in a matrix crystal excited at typical MALDI fluences.
The triplet states of unsubstituted and 5-substituted derivatives of salicylic acid and methyl salicylate, some
of which are used as matrices in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of
macromolecules, were studied in acetonitrile and cyclohexane by transient absorption and time-resolved
luminescence spectroscopy. The results suggest a tautomeric structure of the triplet states of salicylic acid as
well as that of its methyl ester and its 5-hydroxy- and 5-methoxy-substituted derivatives. In this tautomeric
structure, the ortho-hydroxy hydrogen has been transferred to the carbonyl oxygen. No differences were
observed between the triplet−triplet absorption spectra of the acids and the corresponding methyl esters. For
the 5-hydroxy- and 5-methoxy- compounds, evidence for long-lived phototautomers was found. The P-type
delayed fluorescence of methyl salicylate is consistent with the known tautomer fluorescence at 440 nm,
implying a tautomeric structure of the triplet state. Similarly, for the 5-methoxy-substituted compound a unique
delayed fluorescence spectrum, red-shifted relative to the prompt fluorescence spectrum, was observed and
attributed to excimer or tautomer fluorescence. The results presented here contrast with previous reports on
the absence of intramolecular hydrogen atom transfer on the singlet surface of the MALDI matrices 5-methoxy-
and 5-hydroxysalicylic acid and their respective methyl esters. The resulting tautomers and their reaction
products may be relevant for analyte ionization in MALDI.
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