Gas-Phase Ion Spectroscopy of Congo Red Dianions and Their Complexes with Betaine

Congo Red (CR) is an azo dye that is negatively charged in aqueous solutions. Here we report on the intrinsic electronic properties of CR dianions from mass spectroscopy experiments on bare dianions and their complexes with betaine (B). As betaine is a zwitterion, it possesses a large dipole moment and is a good reporter on the sensitivity of CR to microenvironmental changes. Photoexcitation of CR^2– in the visible region resulted in several fragment ions after absorption of at least three photons, with major fragmentation routes due to breakage of one or both C–NN bonds, one azo linkage, and/or the bonds to sulfite. Their yields as a function of excitation wavelength reveal a broad absorption in the visible region with the lowest-energy band located at ∼500 nm. Features are observed with a spacing of ∼1500 cm^–1. One photon was sufficient to dissociate CR^2–·B, and its action spectrum was almost identical to those of CR^2– in accordance with previous findings that a symmetric ion is essentially unaffected by changes in its microenvironment. Electron detachment occurs in the UV with threshold energy of 3.6 ± 0.1 eV for CR^2– and 3.81 ± 0.06 eV for CR^2–·B. Attempts to measure fluorescence from photoexcited CR^2– were unsuccessful

Empirical Calibration of a Cylindrical Ion Trap for Mass-Selected Gas-Phase Fluorescence Spectroscopy

The ion motion in a quadrupole ion trap of hyperbolic geometry is well described by the Mathieu equations. A simpler cylindrical ion trap has also gained significance and has been used by us for fluorescence-spectroscopy experiments. This design allows for the easy replacement of the end-cap with a mesh, enhancing the photon collection. It is crucial to obtain a firm understanding of the ion motion in cylindrical ion traps and their capability as mass spectrometers. We present here an empirical method of calibrating a cylindrical ion trap based on fluorescence detection. This can be done nearly background-free in a pulsed experiment. The ions are located at the center of the trap, where the field is primarily quadrupolar, and here an effective Mathieu description is found through an effective geometry parameter. In spectroscopy experiments, high buffer-gas pressures are needed to efficiently cool the ions, which complicates the ions’ motion and hence their stability. Still, simulations show that the stability diagram closely aligns with the Mathieu diagram, albeit shifted due to collisions. We map the stability diagram for six molecular ions by fluorescence collection from four cations and two anions spanning m/z from 212 to 647. The stability diagram is parametrized through the Mathieu functions with an m/z-dependent effective geometry parameter and a q-dependent shrinkage of the diagram. Based on the calibration, we estimate the mass resolution to be +7/–3 Da for ions with masses in the hundreds of Da

Effect of a Single Water Molecule on the Electronic Absorption by <i>o</i>- and <i>p</i>‑Nitrophenolate: A Shift to the Red or to the Blue?

Many photoactive biomolecules are anions and exhibit ππ* optical transitions but with a degree of charge transfer (CT) character determined by the local environment. The phenolate moiety is a common structural motif among biochromophores and luminophores, and nitrophenolates are good model systems because the nitro substituent allows for CT-like transitions. Here we report gas-phase absorption spectra of <i>o-</i> and <i>p</i>-nitrophenolate·H₂O complexes to decipher the effect of just one H₂O and compare them with ab initio calculations of vertical excitation energies. The experimental band maximum is at 3.01 and 3.00 eV for <i>ortho</i> and <i>para</i> isomers, respectively, and is red-shifted by 0.10 and 0.13 eV relative to the bare ions, respectively. These shifts indicate that the transition has become more CT-like because of localization of negative charge on the phenolate oxygen, i.e., diminished delocalization of the negative excess charge. However, the transition bears less CT than that of <i>m</i>-nitrophenolate·H₂O because this complex absorbs further to the red (2.56 eV). Our work emphasizes the importance of local perturbations: one water causes a larger shift than experienced in bulk for <i>para</i> isomer and almost the full shift for <i>ortho</i> isomer. Predicting microenvironmental effects in the boundary between CT and non-CT with high accuracy is nontrivial. However, in agreement with experiment, our calculations show a competition between the effects of electronic delocalization and electrostatic interaction with the solvent molecule. As a result, the excitation energy of <i>ortho</i> and <i>para</i> isomers is less sensitive to hydration than that of the <i>meta</i> isomer because donor and acceptor orbitals are only weakly coupled in the <i>meta</i> isomer

On the Effect of a Single Solvent Molecule on the Charge-Transfer Band of a Donor–Acceptor Anion

Many biochromophore anions located within protein pockets display charge-transfer (CT) transitions that are perturbed by the nearby environment, such as water or amino acid residues. These anions often contain the phenolate moiety as the electron donor and an acceptor group that couples to the donor via a π-conjugated system. Here we show using action spectroscopy that single molecules of water, methanol, and acetonitrile cause blue shifts in the electronic transition energy of the bare <i>m</i>-nitrophenolate anion by 0.22, 0.22, and 0.12 eV, respectively (uncertainty of 0.05 eV). These shifts are similar to CC2-predicted ones and are in accordance with the weaker binding to the phenolate end of the ion by acetonitrile in comparison with water and methanol. The nitro acceptor group is almost decoupled from the phenolate donor, and this ion therefore represents a good model for CT excitations of an anion. We found that the shift caused by one acetonitrile molecule is almost half of that experienced in bulk acetonitrile solution, clearly emphasizing the important role played by the microenvironment. In protic solvents, the shifts are larger because of hydrogen bonds to the phenolate oxygen. Finally, but not least, we provide experimental data that serve to benchmark calculations of excited states of ion–solvent complexes

On the Influence of Water on the Electronic Structure of Firefly Oxyluciferin Anions from Absorption Spectroscopy of Bare and Monohydrated Ions in Vacuo

A complete understanding of the physics underlying the varied colors of firefly bioluminescence remains elusive because it is difficult to disentangle different enzyme–lumophore interactions. Experiments on isolated ions are useful to establish a proper reference when there are no microenvironmental perturbations. Here, we use action spectroscopy to compare the absorption by the firefly oxyluciferin lumophore isolated in vacuo and complexed with a single water molecule. While the process relevant to bioluminescence within the luciferase cavity is light emission, the absorption data presented here provide a unique insight into how the electronic states of oxyluciferin are altered by microenvironmental perturbations. For the bare ion we observe broad absorption with a maximum at 548 ± 10 nm, and addition of a water molecule is found to blue-shift the absorption by approximately 50 nm (0.23 eV). Test calculations at various levels of theory uniformly predict a blue-shift in absorption caused by a single water molecule, but are only qualitatively in agreement with experiment highlighting limitations in what can be expected from methods commonly used in studies on oxyluciferin. Combined molecular dynamics simulations and time-dependent density functional theory calculations closely reproduce the broad experimental peaks and also indicate that the preferred binding site for the water molecule is the phenolate oxygen of the anion. Predicting the effects of microenvironmental interactions on the electronic structure of the oxyluciferin anion with high accuracy is a nontrivial task for theory, and our experimental results therefore serve as important benchmarks for future calculations