Ionization Methods IV:Bbd tMALDIdBombardment, MALDI and ElementalCU- Boulder Lecture IVJ. Kimmel / J. L.JimenezFall 2009•Physical states of sample•Chromatographic coupling•Sample/matrix complexity•Destruction of matrix•Selectivity•Ionization efficiency •Coupling / transmission to MS •Molecular mass and/or structural elucidation•Degree of FragmentationEI and CI are methods for molecular analysisEI and CI are methods for molecular analysis of gas phase sampleAPCI and ESI: molecular analysis of liquid phase Now, desorption techniques that allow molecular analysis from static liquid or solid matrixFast Atom Bombardment (FAB) and Secondary Ion Mass Spectrometry (SIMS)From WatsonDesorption techniques. Analyze the ions emitted when a surface is irradiated with an energetic beam of neutrals (FAB) or ions (SIMS).Static SIMS• Low current (10-10A cm2) of keV primary ions (Ar+, Cs+ ..) impact the solid analyte surface•Low probability of area being struck by mulitiple ions;less than 1/10 ofLow probability of area being struck by mulitiple ions; less than 1/10 of atomic monolayer consumed• Primary ion beam focused to less than 1 um enables high resolution mapping – Often pulsed beam + TOFMS• Sensitive technique for the ID of organic molecules•Spectra show high abundance of protonated or cationizedmolecularSpectra show high abundance of protonated or cationized molecular ions.• Yields depend on substrate and primary beam; as high as 0.1 ions per incident ion. Elemental yields vary over many orders of magnitude.Producing Primary BeamFrom de HoffmannSIMS iiPi B ftlSIMS primary ions are produced, e.g., as Cs atoms vaporize through a porous tungsten plug. Primary Beam of neutralsproduced by ionizing and accelerating compound into charge exchange collision with neutral. e.g.:Ar+rapid+ Arslow→ Ar+slow+ ArrapidFAB and liquid-SIMS• Sample is dissolved in non-volatile liquid matrix and bombarded with beam of neutrals (FAB) or ions • Shock wave ejects ions and molecules from solution. Generally eject ions that already exist in solutionthat already exist in solution.• Presence of charge (SIMS vs FAB) has little effect on the desorption process. Neutrals used out of convenience for coupling to some instruments.• Use of liquid allows high primary ion currents while maintaining molecular ions – Solution presents mobile, constantly renewed surface to beam• Disadvantage: substantial background of matrix (e.g., Glycerol) ions and matrix adducts. • Flow FAB: – Continuous flow of liquid into mass spectrometer at rate of 1 – 20 uL/min– Allows more use of more volatile solvent (eg, water, methanol, or acetonitrile)– Can be coupled to separationMatrix Assisted Laser Desorption Ionization (MALDI)• Analyte molecules are embedded in a crystalline matrix composed of a low molecular weight organic species. •Dried mixture is struck with a short intense laserDried mixture is struck with a short, intense laser pulse having a wavelength that is strongly absorbed by the matrix (often UV). • Rapid heating of matrix causes sublimation and expansion into gas phase. Intact analyte molecules carried with little internal energy.• Most widely accepted ionization mechanism is gas-phaseproton transferphase proton transfer. • Efficient, “soft,” and relatively universal(wavelength independent of analyte). Matrix isolates analyte molecules, preventing clusters.• Allows analysis of large (100s of kDa) intact biopolymers (2002 Nobel Prize)Image from: Univ. of Bristolhttp://www.chm.bris.ac.uk/ms/theory/maldi-ionisation.htmlExample of MALDI Data•Spectra contain mostly single-charged ions.•Fragmentation due to excess energy imparted on analyte during DI process is possible (Prompt, Fast, or Post Source)From De Hoffmann•Optimized conditions determined empirically.Two Step IonizationLaser Pulse (nanoseconds)Relative density of plume (microseconds)From: Reactive species see rapidly changing environment – most important is the density of species.Primary matrix ions created by laser pulse (blue)Many microseconds to reach collision-free density. Secondary reactions during this time. Red plot shows a smooth transition from a solid to a gas = desorption.http://www.rknochenmuss.ch/MALDI/Two Step IonizationTLaserTimeAngstroms•Initial = smooth transition (desorption)•Heating causes pressure wave. Compression followed by a rebound, expansive wave. •Subsurface nucleation, followed by phase explosion, yielding dense mixture of clusters, droplets and chunks.•Complicated reactive environment, but only those things that become gaseous are of interest.AngstromsFrom: http://www.rknochenmuss.ch/MALDI/Pooling Events•Material is heated in a few nanosec – faster than vaporization takes place•Initial period is thus high energy and high density. PRIMARY ionization takes place at this point (before vaporization)•Ionization potentials of most matrices are above 1 and 2-photon energies of•Ionization potentials of most matrices are above 1 and 2-photon energies of typical laser•Energy is concentrated on molecule by 2 sequential pooling events. •Excitation hops through pi-electron systems of dense matrix This process produces the bulk of the primary ions.From: http://www.rknochenmuss.ch/MALDI/Secondary Ionization•Density changes slowly in plume and many collisions occur. •Analyte ions are derived from primary matrix ions by one or more of 3 reactions (M=matrix A=analyte):(M=matrix, A=analyte):Proton transfer MH++ A ↔ M + AH+and (M-H)-+ A ↔ M + (A-H)-Electron transfer m++ A ↔ m + A+and m-+ A ↔ m + A-Cation transfer e.g. mNa++ A ↔ m + ANa+•Many collisions yields local thermal equilibrium, leaving thermodynamically yy q gyymost favorable ions.•Despite complex environment, final collisions for any analyte is generally bimolecular with m. And aggregates, etc, can be ignored.Thermodynamically Favored Product•Data show varied M/A ratio•Various ion types interconvert in plume, with charge flowing to thermodynamically favored analyte ion•Analytescan also•Analytescan also suppress each otherMALDI-TOF Mass SpectrometerFrom BarkerMALDI conveniently coupled to ToF-MS1. Pulsed source, pulsed analyzer2. Large m/z, large mass rangeAP MALDI•Allows use of matrices that are not vacuum compatible (liq ids)(liquids)•Softer than standard, because of fast thermalization•Advances have improved sensitivity in modernsensitivity in modern