measurable change in the light intensity. Typically, either electrospray ionisation
(ESI) [1] or matrix assisted laser desorption ionisation (MALDI) [2, 3] are used for
the production of biomolecular ions, and ion currents may be 0.1 pA. Ions are
accumulated in an ion trap for say 0.1 s (experiment with a 10-Hz repetition rate),
which results in a total charge of 10
À14 C corresponding to about 10
5 singly charged
ions in a volume of maybe 1 mm
3 . For comparison, in a solution with the same
volume and a sample concentration of 0.1 mM, the number of chromophore ions is
10
13 , a difference of eight orders of magnitude! Clearly, alternative methods are
required to measure the absorbance by thin gas-phase ion targets.
One such method is called action spectroscopy, as the measurement requires an
observable “action”, e.g. dissociation, to determine an absorption spectrum: When
gas-phase ions are excited to a higher energy level through photon absorption,
several different de-excitation channels are possible; for example, dissociation from
the excited-state potential energy surface, internal conversion to a hot ground state
and subsequent dissociation, electron detachment (anions) or fluorescence. In
action spectroscopy, the daughter products formed after dissociation or electron
detachment are monitored at each wavelength, and an action spectrum compiled as
the magnitude of photoproducts correlates with the amount of absorption. These
spectra are identical to absorption spectra if fluorescence is not a possible pathway
or the fluorescence quantum yield is independent of excitation wavelength. In the
following, different experimental setups used for action spectroscopy are described.
3.1.1 Electrostatic Ion Storage Rings
Electrostatic storage rings store ions by using a number of electrostatic deflectors
such as parallel plates, cylindrical deflectors and quadrupoles to deflect the ions into
different straight sections along a closed path. Ions circulate in the ring and can be
monitored over time and probed while in straight sections. Numerous different
configurations are possible depending on the number of sections required. As an
example, in the ELectrostatic Ion Storage ring in Aarhus (ELISA) ions are deflected
using two sets of three deflectors each consisting of a set of parallel plates which
deflect the ions 10
out of one straight section, a cylindrical deflector which
reorients the ions 160
, and a second set of parallel plates which deflect the ions
10
into the other straight section (Fig. 3.1).
In ELISA biomolecule ions are produced using electrospray ionisation,
accumulated in a 22-pole ion trap and thermally equilibrated by collisions with a
helium buffer gas therein (Fig. 3.2). They are subsequently accelerated as a bunch
to kinetic energies of 22 keV, and the desired ions selected using a bending magnet.
Following injection into the ring, the lifetimes of ions can be monitored over time
by detecting one of the following:
1. The production of neutral fragments after dissociation from either vibrationally
excited ions or from ions that collide with the residual gas (collision induced
dissociation (CID)). After dissociation, ionic fragments will be deflected along
an incorrect path and will not be stored in the ring, while neutral products will be
22
J.A. Wyer
(ESI) [1] or matrix assisted laser desorption ionisation (MALDI) [2, 3] are used for
the production of biomolecular ions, and ion currents may be 0.1 pA. Ions are
accumulated in an ion trap for say 0.1 s (experiment with a 10-Hz repetition rate),
which results in a total charge of 10
À14 C corresponding to about 10
5 singly charged
ions in a volume of maybe 1 mm
3 . For comparison, in a solution with the same
volume and a sample concentration of 0.1 mM, the number of chromophore ions is
10
13 , a difference of eight orders of magnitude! Clearly, alternative methods are
required to measure the absorbance by thin gas-phase ion targets.
One such method is called action spectroscopy, as the measurement requires an
observable “action”, e.g. dissociation, to determine an absorption spectrum: When
gas-phase ions are excited to a higher energy level through photon absorption,
several different de-excitation channels are possible; for example, dissociation from
the excited-state potential energy surface, internal conversion to a hot ground state
and subsequent dissociation, electron detachment (anions) or fluorescence. In
action spectroscopy, the daughter products formed after dissociation or electron
detachment are monitored at each wavelength, and an action spectrum compiled as
the magnitude of photoproducts correlates with the amount of absorption. These
spectra are identical to absorption spectra if fluorescence is not a possible pathway
or the fluorescence quantum yield is independent of excitation wavelength. In the
following, different experimental setups used for action spectroscopy are described.
3.1.1 Electrostatic Ion Storage Rings
Electrostatic storage rings store ions by using a number of electrostatic deflectors
such as parallel plates, cylindrical deflectors and quadrupoles to deflect the ions into
different straight sections along a closed path. Ions circulate in the ring and can be
monitored over time and probed while in straight sections. Numerous different
configurations are possible depending on the number of sections required. As an
example, in the ELectrostatic Ion Storage ring in Aarhus (ELISA) ions are deflected
using two sets of three deflectors each consisting of a set of parallel plates which
deflect the ions 10
out of one straight section, a cylindrical deflector which
reorients the ions 160
, and a second set of parallel plates which deflect the ions
10
into the other straight section (Fig. 3.1).
In ELISA biomolecule ions are produced using electrospray ionisation,
accumulated in a 22-pole ion trap and thermally equilibrated by collisions with a
helium buffer gas therein (Fig. 3.2). They are subsequently accelerated as a bunch
to kinetic energies of 22 keV, and the desired ions selected using a bending magnet.
Following injection into the ring, the lifetimes of ions can be monitored over time
by detecting one of the following:
1. The production of neutral fragments after dissociation from either vibrationally
excited ions or from ions that collide with the residual gas (collision induced
dissociation (CID)). After dissociation, ionic fragments will be deflected along
an incorrect path and will not be stored in the ring, while neutral products will be
22
J.A. Wyer