that the electronic transition becomes more CT like (decoupling of the overlap
between donor and acceptor states); CT transitions are often characterised by low
transition energies and low oscillator strengths. In the extreme case of no overlap
between the HOMO and LUMO orbitals (Fig. 2.3), the excitation energy is simply
the difference between the ionisation energy of the donor and the electron affinity of
the acceptor. Intermediate transitions are particularly difficult to account for theoretically. A picture is emerging where the situation in vacuo seems much closer to
that of the chromophore within the protein pocket than that of the chromophore in
bulk aqueous solution, at least when it comes to the chromophore’s electronic
structure. Still, however, both experimental and theoretical data are needed to
explain each individual chromophore case.
2.2
New Aspects and Challenges
Despite the impressive amount of work that has been done in the field, some of it
being presented in Chaps. 3–11, it is far from dying out, and more groups worldwide than ever are working on spectroscopy of biomolecular ions. Indeed, there are
many interesting new aspects and certainly also challenges to address in the future.
Some of them will be discussed in the following.
One frequent complication of the experiments is the presence of multiple
isomers in the ion beam that all contribute to the electronic absorption spectrum,
to the deexcitation scheme, or even to the dissociation pattern if they do not quickly
interconvert. The implementation of an ion mobility device [23] right after the ion
source could in many cases solve this issue as this would separate ions with
different structures in time due to different drift times through the carrier buffer
gas (Fig. 2.7). The combination of mass spectrometry (separation by mass) and ion
mobility spectrometry (separation by geometrical structure) indeed seems a very
promising and attractive direction for the future. A very recent paper by Bieske and
co-workers [24] nicely demonstrates the strength of an ion mobility spectrometer
combined with action spectroscopy for the study of the photoisomerisation of
molecular ions. Here absorption reveals itself as a geometry change of the molecular ion resulting in a different drift time through the mobility cell! This issue of
photoisomerisation is needless to say a highly relevant issue for many protein
biochromophores such as the cis-trans isomerisation of retinal.
An action spectrum is often taken to represent the gas-phase absorption spectrum. This is in many cases a valid and reasonable assumption (see for example
Chap. 3 by Wyer). However, when fluorescence is important, this may not be
justified as the fluorescence quantum yield could depend on the excitation wavelength. An ambitious task would be to establish the fluorescence quantum yields for
each excitation wavelength to correct for such variations. Likewise, in the
photoisomerisation experiment, the isomerisation process may be wavelength
dependent, and the absorption spectrum is then modulated by the photoisomerisation probability. Finally, in experiments relying on dissociation, finite time
windows for measuring fragmentation can skew the action spectra to either the
2 Introduction and New Aspects
15
between donor and acceptor states); CT transitions are often characterised by low
transition energies and low oscillator strengths. In the extreme case of no overlap
between the HOMO and LUMO orbitals (Fig. 2.3), the excitation energy is simply
the difference between the ionisation energy of the donor and the electron affinity of
the acceptor. Intermediate transitions are particularly difficult to account for theoretically. A picture is emerging where the situation in vacuo seems much closer to
that of the chromophore within the protein pocket than that of the chromophore in
bulk aqueous solution, at least when it comes to the chromophore’s electronic
structure. Still, however, both experimental and theoretical data are needed to
explain each individual chromophore case.
2.2
New Aspects and Challenges
Despite the impressive amount of work that has been done in the field, some of it
being presented in Chaps. 3–11, it is far from dying out, and more groups worldwide than ever are working on spectroscopy of biomolecular ions. Indeed, there are
many interesting new aspects and certainly also challenges to address in the future.
Some of them will be discussed in the following.
One frequent complication of the experiments is the presence of multiple
isomers in the ion beam that all contribute to the electronic absorption spectrum,
to the deexcitation scheme, or even to the dissociation pattern if they do not quickly
interconvert. The implementation of an ion mobility device [23] right after the ion
source could in many cases solve this issue as this would separate ions with
different structures in time due to different drift times through the carrier buffer
gas (Fig. 2.7). The combination of mass spectrometry (separation by mass) and ion
mobility spectrometry (separation by geometrical structure) indeed seems a very
promising and attractive direction for the future. A very recent paper by Bieske and
co-workers [24] nicely demonstrates the strength of an ion mobility spectrometer
combined with action spectroscopy for the study of the photoisomerisation of
molecular ions. Here absorption reveals itself as a geometry change of the molecular ion resulting in a different drift time through the mobility cell! This issue of
photoisomerisation is needless to say a highly relevant issue for many protein
biochromophores such as the cis-trans isomerisation of retinal.
An action spectrum is often taken to represent the gas-phase absorption spectrum. This is in many cases a valid and reasonable assumption (see for example
Chap. 3 by Wyer). However, when fluorescence is important, this may not be
justified as the fluorescence quantum yield could depend on the excitation wavelength. An ambitious task would be to establish the fluorescence quantum yields for
each excitation wavelength to correct for such variations. Likewise, in the
photoisomerisation experiment, the isomerisation process may be wavelength
dependent, and the absorption spectrum is then modulated by the photoisomerisation probability. Finally, in experiments relying on dissociation, finite time
windows for measuring fragmentation can skew the action spectra to either the
2 Introduction and New Aspects
15