The ionization mechanisms in matrix-assisted laser desorption/ionization (MALDI) remain poorly understood. We have begun a program of study aimed at determining the properties of matrix molecules which make them suitable for MALDI. Initial results are presented here for one of the most widely used matrix materials, 2,5-dihydroxybenzoic acid (DHB). Spectroscopy of free DHB molecules in a molecular beam shows that the photoionization energy is much lower than expected from semiempirical calculations, only 8.05 eV, yet still not accessible with two nitrogen laser photons (7.36 eV). No evidence is found for labile protons in the first excited state. This is in spite of structural similarity with salicylates where excited state proton transfer from the 2-hydroxy group occurs. Conventional excited state proton transfer from single DHB molecules to analytes is thus deemed unlikely. On the other hand, a two-step reaction taking place via decarboxylated DHB (hydroquinone) is shown to be a potential analyte protonation mechanism. The conclusion is reached that single matrix molecules are probably not the primary ion generators in UV MALDI with this matrix. This is consistent with proposed models which require dimers or larger aggregates for ion generation. The photo/thermal combined ionization model of Allwood, Dyer and Dreyfus (Rapid Commun. Mass Spectrom. 11, 499 (1997)) is updated with measured physical parameters for DHB, and extended to include 2-center energy pooling mechanisms. #

As recently shown for positive ions (Rapid Commun. Mass Spectrom. 10, 871 (1996)), small to moderate-sized analyte ions (`20 000 Da) can fully suppress all matrix signals in matrix-assisted laser desorption/ionization (MALDI) mass spectra at appropriate matrix:analyte mixing ratios. The technique is useful for removing matrix interferences and led to a model for primary ion formation mechanisms. We here extend the method to negative ions, and investigate the effects of analyte type and size on the matrix suppression concentration range. For a given matrix/analyte pair, suppression was observed in either positive-or negative-ion mode, but not both. Some matrices do not show the effect at all, which is attributed to unfavourable thermodynamics. The analyte concentration where suppression appears is correlated with the analyte molecular weight and supports the proximity requirement of the model. The dependence of matrix signals on analyte concentration is very similar in both modes, and may have implications for the role of plume reactions at low analyte concentrations. Delayed extraction was found to extend matrix suppression to lower analyte concentrations. Lack of homogenous matrix/analyte co-crystallization at low analyte concentrations is shown to prevent the appearance of matrix suppression or to modify the shape of the concentration curves in some cases. Evidence is presented for the activity of electronic excitations in IR as well as in UV MALDI.

Resonant two-photon ionization (R2PI) and fluorescence spectra of the 1-naphthol·H2O and 1-naphthol-d 1·D2O cluster are reported and interpreted with emphasis on intracomplex vibrational energy redistribution (IVR) and vibronic coupling. The analysis included an ab initio normal-coordinate calculation of both complexes at the 6-31G (d,p) level. In S1 ← S0 R2PI spectra, the intermolecular modes were found to be very weak; only one such S1 vibration was observed at 57 cm-1 (54 cm-1 for 1-naphthol-OD·D2O). At excitations less than 400 cm-1 above the S1← S0 origin, the fluorescence spectra retain a simple 1-naphthol-like pattern. At higher excess energies, the fluorescence exhibits complex structure due to emission from both relaxed and unrelaxed S1 vibrational states, showing efficient coupling through the hydrogen bond. This restricted IVR behavior changes to dissipative IVR at about 834 cm-1 above the S1 ← S0 origin. Exceptions to general IVR are observed. The S1 + 403 cm-1 level is a restricted IVR case, while the nearby S1 + 400 cm-1 level undergoes nearly dissipative IVR. On the other hand, a nearly “frozen” mode is observed at 553 cm-1 above the S1← S0 origin. By use of the ab initio vibrational analysis and lowest-order anharmonic coupling, the IVR-active vibrational “bath” states can be predicted, enabling tentative assignment of much of the relaxed fluorescence structure. Ion-dip spectroscopy shows that IVR in the electronic ground state may have a higher onset threshold than in the S1 by ≥130 cm-1. Exciting one quantum of the 57 cm-1 intermolecular mode results in a decreased S0 IVR threshold. The strong vibronic S1−S2 coupling earlier observed for vibrations at S1 + 410 and S1 + 414 cm-1 in free 1-naphthol was found to be partially inhibited in 1-naphthol·H2O but still significant in 1-naphthol-OD·D2O. This is attributed to harmonic mode mixing. In contrast, vibronic coupling becomes active in the S1 + 292 cm-1 level of 1-naphthol·H2O.

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