Page 263 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

P1: GTM/GRL P2: GNH Final Pages
Encyclopedia of Physical Science and Technology EN009N-406 July 18, 2001 23:32

Mass Spectrometry 151

As the solvent emerges from the charged capillary, it mass spectra, and even spatially resolved mass spectral
first forms a cone (called a Taylor cone) that results as maps of a surface. Organic and biological molecules de-
the droplet adopts a shape to mimimize Coulombic re- compose under such intense irradiation, and despite much
pulsions between the charges on the surface of the liq- early work in the area, suitable conditions for successful
uid. The initially formed cone then dissociates into small direct analysis were not found. In MALDI, a matrix is used
droplets, each now isolated in the gas phase (and still to moderate on a molecular level the energy deposited at
at atmospheric pressure), and each carrying an excess thesurface,andpromoteionizationofthesamplemolecule
charge on its surface. Desolvation involves the loss of without decomposition or excessive dissociation. In prac-
neutral solvent molecules from the droplet, and proceeds tice, the sample is mixed in solution with a large excess
rapidly at atmospheric pressure. As the droplet decreases of the matrix (typically a smaller molecule chosen for
in size, the charge density increases until an instabil- high UV absorbance, appropriate volatility, and ability to
ity limit is reached, and the droplet dissociates into still promote ionization), and an aliquot of a few microliters
smaller highly charged droplets. Residual solvent quickly volume is deposited on an inert surface to co-crystallize.
evaporates, leaving only the charged ions themselves to be The surface is irradiated with short (10–20 nsec) pulses
transferred into the mass spectrometer. The ionic popula- of laser light at 337 nm; a tight focus on the surface is
tion within a microdroplet, and then a nanodroplet, is not not necessary. The irradiance power achieved is approxi-
2
6
known explicitly. The charge imposed on the droplet as it mately 10 W/cm .
departs the needle must be carried by a chemical species in The ions observed in the MALDI mass spectrum (they
the solution. If the droplet is positively charged, the charge have been termed the “survivor” ions) are the minority
carriers will be predominantly protons. As the droplet de- within all the species that leave the surface as a result of the
creases in size, the “pH” rises exponentially (pH is not laser beam irradiation, comprising perhaps 0.01% of the
an accurate description of the situation since there is no desorbed/ablated/expelled species (Zenobi and Knochen-
equilibrium). As the droplet decreases in size, the protons muss, 1999). Since the presence of the matrix is requisite
are forced to ionize the sample molecules. Protonation, for MALDI, investigators quite logically seek to establish
and in fact multiple protonation, is commonly observed. the role of the matrix by changes to its chemical nature,
Key aspects of the ESI process are the formation of with consequent changes to its intrinsic proton affinity,
multiple charged ions of the sample molecule, and mini- its absorbance spectrum, its ionization potential, and its
mum fragmentation of these molecular ions. Positive ions crystal structure. While the analyte-to-matrix ratio may
of the general form (M + nH) n+ are formed by multiple be controlled on a macroscopic level, it varies across a
protonationoflargerbiomolecules(molecularmassisdes- broad range on the microscopic level, and will also vary
ignated by M) such as peptides and proteins. One effect spot to spot. The incident laser beam interrogates a surface
of multiple charging is to bring multiply charged higher on which crystals of different size and different properties
mass molecules within the mass range of commonly used are dispersed. The microscopic morphological effects that
mass spectrometers, since the mass analysis is actually a result from a change in gross sample preparation proce-
m/z measurement. Further, since M is constant between dure are uncharacterized. The simple fact of the matter is
the series of peaks observed as adjacent multiply charged that MALDI works amazingly well under a wide variety
ions, the multiple measurements of mass of these ions of conditions, and it is perhaps not necessary to control
constitute a series of simultaneous equations that can be all of them rigidly. It has become recognized that in ad-
solved to determine M, the molecular mass, to a pre- dition to the direct desorption of species from the laser-
cision of ±0.005%. This mass measurement capability irradiated surface, secondary ionization processes occur
was not an improvement on exiting methodology, but was in the selvedge above the surface. This is a reiteration of
a totally new capability, without precedent and without the concept of the selvedge introduced to explain reactions
competition from other analytical methods. This unique- in secondary ion mass spectrometry and desorption ion-
ness makes accurate prediction of future applications ization techniques in general (Cooks and Busch, 1983).
and developments in mass spectrometry challenging and Reactions that transfer, preserve, or dissipate charge de-
exciting. termine what ions finally survive to form the MALDI mass
spectrum. Proton transfer reactions that may involve the
matrix form protonated molecules. Alkali cations at the
4. Matrix-Assisted Laser Desorption Ionization
surface as impurities can form cationized species. In a pro-
A pulsed laser can be used to deposit a very large amount cess reminiscent of ESI, multiply charged species can be
of energy into a small spot in a short time. This energy formed, but the high population of free electrons, and the
causes ionization, and if the desired ions are atomic ions, high mobility of electrons within the selvedge, promotes
then laser desorption proves a useful method for creating reduction processes that reform the singly charged ions.