Mass Spectrometry

1. Mass Spectrometry 2012 Fall version dslee@swu.ac.kr

2. Basic theory of mass spectrometry The mass spectrometer(MS) is an instrument that serves for establishment of the molecular weight and structure of both inorganic and organic compounds, and the identification and determination of analytes in complex mixtures. The MS is an instrument capable of producing a beam of ions from sample under investigation, separating these ions according to their mass-to charge (m/z) ratios and recording the relative abundances of the separated ion species as a mass spectrum. The ion-currents corresponding to the different species are amplified and either displayed on an oscilloscope or a chart-recorder, or are stored in a computer. The peak intensities are plotted as ordinates, in arbitrary units or normalized with respect to the most important peak, which is assigned a value of 100.

3. 100 128 80 a 60 Relative abundance (%) 40 102 20 77 64 c c c 51 b b b b 0 50 100 150 Mass to charge ratio m/z Mass spectrum of CO2. Note that the molecular ion appears at m/z = 44 (C = 12, O = 16). Frag-ment ions appear at m/z values of 28, 16, and 12. These correspond to CO+, O+, and C+, resp-ectively. The mass spectrum of naphthalene with electron impact ionization by 70 eV electrons. a, molecular ion and base peak(C10H+8, 100%); b, 13C isotope peak; c, fragment ion peaks. ,

4. Introduction to mass spectrometry Mass spectrometers use the difference in mass-to-charge ratio (m/e) of ionized atoms or molecules to separate them from each other. Mass spectrometry is therefore useful for quantitation of atoms or molecules and also for determining chemical and structural information about molecules. Molecules have distinctive fragmentation patterns that provide structural information to identify structural components. All commonly used mass analyzers use electric and magnetic fields to apply a force on charged particles (ions). The relationship between force, mass, and the applied fields can be summarized in Newton's second law and the Lorentz force law: F = ma (Newton's second law) F= e(E+ v× B) (Lorentz force law) where F is the force applied to the ion, m is the mass of the ion, a is the acceleration, eis the ionic charge, E is the electric field , v B is the vector cross product of the ion velocity and the applied magnetic field.

5. From Newton's second law, it is apparent that the force causes an acceleration that is mass-dependent, and the Lorentz force law tells us that the applied force is also dependent on the ionic charge. Therefore, it should be understood that mass spectrometers separate ions according to their mass-to-charge ratio (m/z) rather than by their mass alone. • The general operation of a mass spectrometer is: • create gas-phase ions • separate the ions in space or time based on their mass-to-charge ratio • measure the quantity of ions of each mass-to-charge ratio • The ion separation power of a mass spectrometer is described by the resolution R, which is usually defined as:  • where m is the ion mass and delta m is the difference in mass between two resolvable peaks in a mass spectrum.

6. Mass Spectrometry Theory In mass spectrometry, a small sample of a chemical compound is vaporized, bombarded with high energy electrons to ionize the sample, and the ions produced are detected based on the charge to mass ratio of the ions. Ionization process in mass spectrometry.

7. Several different types of ions are produced in this process. If the compound loses only one electron, then a molecular ion, having the same mass as the original compound, is produced. This molecular ion is called the M+ ion (and it gives us the exact molecular weight of the compound). The stream of high energy electrons is sufficiently powerful to break chemical bonds in the molecule, producing a series of molecular fragments. These positively charged fragments are also detected by the instrument, producing the mass spectrum. Organic chemical compounds will fragment in very specific ways depending on what functional groups are present in the molecule. Analysis of the fragmentation pattern can lead to the determination of the structure of the molecule. Fragments produced by benzamide.

8. the analyte (A-B-C) undergoing ionization and fragmentation • the charged fragments (A+ B+ C+) being separated by mass • the fragments which are focused on the mass filter's exit slit passing into the detector • and the charged ions being detected.

9. Instrumentation Components of a mass spectrometry 1) Inlet system : introduce a very small amount of sample ( micromole or less) into the mass spectrometer, where its components are converted to gaseous ions. 2) Ion source : converts the components of a sample into ions by bombardment with electrons, ions, molecules, or photon. 3) Mass analyzer : separates the analyte ions according to their m/z ratios. 4) Detector : converts the beam of ions into an electrical signal(currents) ; the detector output can be displayed or stored, to yield the mass spectrum. 5) Electronics of power supply and control of the systems. 6) Vacuum systems : maintain low pressures ( 10–5 to 10–8 torr); rotary vacuum oil pump, diffusion pump, turbomolecular pump.

10. Sample introduction Interfacing Ion Source Analyzer Detector Data System • EI • CI • FAB • MALDI • ESI • APCI • Thermospray • Particle Beam • Quadrupole • Sector • TOF • FT-ICR • Ion Trap • Library • GC • LC • SFC • IC • CE • DLI • ICP • Solid probe Vacuum System: DP or TMP • MS/MS – Q/TOF, TOF/TOF • HRMS • LRMS Mass Spectrometer Components 10–5 ~ 10–8torr

11. Schematic diagram of mass spectrometer.

12. Vacuum system • Oil rotary pump : 50 ~ 200 ml/min • Turbomolecular pump : no cooling water, 10 min. • Oil diffusion pump : cooling water, 40~50 min. 600 ~ 1000 l/sec • Vacuum in MS 10-6 to 10-5 Torr • - Increase sensitivity • - avoid ion-molecule reaction • - collision free ion trajectories • - avoid background interference • Vacuum System: • - Pumps must be maintained ; Ultra-clean ; Vacuum regulation and maintenance

13. High vacuum Ion Source Mass Analyzer Detector Data system Interface to vacuum High vacuum Interface to vacuum Mass Analyzer Detector Data system Ion Source MS Configuration for Ionization EI API, APCI, ESI

14. Sample introduction For EI and CI there are four main options Heated reservoir septum inlet for pure gases or volatile liquids. Basically this is a heated reservoir (~200oC) with a small restriction ‘bleed’ into the ion source. Sample is injected into the reservoir through a septum. Direct insertion probe for volatile solids. Sample is loaded into a quartz tube at the end of a probe and inserted directly into the ion source. The end of the probe can then be heated, if required, up to temperatures in excess 400oC. Gas chromatograph (GC) for volatile, non-polar mixtures. The mixture is injected into the top of the GC column the end of which is passed, via a heated interface, directly into the ion source. The components of the mixture separate out during their passage through the column and enter the ion source sequentially. Particle Beam interface for semi-volatile compounds that are amenable to EI and CI. This is a useful LC/MS interface that can also be used for rapid introduction of samples by flow injection. Samples are dissolved in a suitable solvent and the solution is introduced into the mass spectrometer using a suitable (e.g. HPLC) pump. The liquid is nebulised with helium gas to form an aerosol of solvent droplets. The stream of liquid droplets passes through a desolvation chamber and then a series of nozzles and skimmers that remove the solvent and helium, allowing a stream of solid particles (the sample) to enter the EI/CI ion source.

15. Chromatographic inlet systems A> Interfacing GC with MS 1. Molecular jet separator (all-glass) : Ryhage, 1966 2. Membrane separator : Llewellyn, 1966 3. Effusion separator : Watson-Bieman, 1964 4. Open split coupling 5. Capillary direct interface B> LC-MS interface 1. Moving belt wire interface 2. Thermospray 3. Atmospheric pressure inlet (API) 4. Electrospray 5. Particle beam 6. Dynamic FAB GC-MS : Thermal vaporization of the analyte, separation, transfer of analyte to the MS, ionization and detection GC Transfer line MS Injection Separation Ionization Ion filtering Detection Transfer LC-MS : Separation, nebulization of the analyte, transfer of ions to the MS, and detection LC Interface MS Injection Separation Transfer Ionization Ion filtering Detection

16. Ionizer In the GC-MS discussed in this introduction, the charged particles (ions) required for mass analysis are formed by Electron Impact (EI) Ionization.  The gas molecules exiting the GC are bombarded by a high-energy electron beam (70 eV).  An electron which strikes a molecule may impart enough energy to remove another electron from that molecule.  Methanol, for example, would undergo the following reaction in the ionizing region:         CH3OH + 1 e  CH3OH+. + 2 e note:  the symbols +. indicate that a radical cation (radical ion) was formed EI Ionization usually produces singly charged ions containing one unpaired electron.  A charged molecule which remains intact is called the molecular ion.  Energy imparted by the electron impact and, more importantly, instability in a molecular ion can cause that ion to break into smaller pieces (fragments).  The methanol ion may fragment in various ways, with one fragment carrying the charge and one fragment remaining uncharged.  For example:          CH3OH+. (molecular ion)  CH2OH+(fragment ion) + H.(or)   CH3OH+. (molecular ion)  CH3+(fragment ion) + .OH http://www.chem.arizona.edu/massspec/intro_html/intro.html

17. Ionization techniques - EI (electron impact) - CI (chemical ionization) - FAB (fast atom bombardment) - ESI (electrospray ionization) - MALDI (matrix assisted laser desorption ionization) - APCI (atmospheric pressure chemical ionization)

18. Ionizationmethod TypicalAnalytes SampleIntroduction MassRange MethodHighlights Electron Impact (EI) Relativelysmallvolatile GC orliquid/solidprobe to1,000Daltons Hard methodversatileprovidesstructure info Chemical Ionization (CI) Relativelysmallvolatile GC orliquid/solidprobe to1,000Daltons Soft methodmolecular ionpeak [M+H]+ Electrospray (ESI) PeptidesProteinsnonvolatile LiquidChromatographyor syringe to200,000Daltons Soft methodions oftenmultiplycharged Fast Atom Bombardment (FAB) CarbohydratesOrganometallicsPeptidesnonvolatile Sample mixedin viscousmatrix to6,000Daltons Soft methodbut harderthan ESI orMALDI Matrix Assisted Laser Desorption(MALDI) PeptidesProteinsNucleotides Sample mixedin solidmatrix to500,000Daltons Soft methodvery highmass Sample introduction / ionization method:

19. Electron Ionization (EI)  Theory Electrons are produced by thermionic emission from a tungsten or rhenium filament. A typical filament current is 1x10-4Amps. These electrons leave the filament surface and are accelerated towards the ion source chamber which is held at a positive potential (equal to the accelerating voltage). The electrons acquire an energy equal to the voltage between the filament and the source chamber - typically 70 electron volts (70 eV). The electron trap is held at a fixed positive potential with respect to the source chamber. A proportion of the electron beam will strike the electron trap producing the trap current. This is used as a feedback circuit to stabilize the electron beam. A permanent magnet is positioned across the ion chamber to produce a magnetic flux in parallel to the electron beam (see figure). This causes the electron beam to spiral from the filament to the trap, increasing the chance and efficiency of analyte ionization. Gaseous analyte molecules are introduced into the path of the electron beam where they may be ionized by electronic interactions with this electron beam. Ionization can also be brought about by direct impaction of an electron with an analyte molecule. The positive ion repeller voltage and the negative excitation voltage work together to produce an electric field in the source chamber such that ions will leave the source through the ion exit slit. The ions are directed through the various focussing and centering lenses and are focussed onto the source exit slit. The ions can then be mass analysed. http://www-methods.ch.cam.ac.uk/meth/ms/theory/eims.html

20. Schematic of a Kratos Analytical Electron Ionization source (as used on the MS890). http://www-methods.ch.cam.ac.uk/meth/ms/theory/eims.html http://masspec.scripps.edu/information/intro/chapter3.html#3.2.1.

21. Mechanisms of ion formation • Consider the ionization of the analyte species AB: • AB + e-* A+ + B- + e- • AB + e-* A+ + B° + 2e- • AB + e-* [AB+°*] + 2e- followed by [AB+°*]  AB+° • AB + e-* [AB2+*]" + 3e- followed by [AB2+*]"  A+ + B+ • - very low abundance • AH + e-* AH* + e-followed by AH* + AH  [AH+H]+ + A- • - 'self chemical ionization' • Notes: • Species with a * superscript are in high energy sates. Species with a ° superscript are radicals. • Species with a " superscript are short lived intermediates which are not seen in the spectra. • 1 and 2 are the highest abundance and are termed instantaneous fragmentation. This is the reason why EI is considered a hard ionization process. • 3 is fairly high abundance and is the process responsible for the molecular ion formation. Unfortunately the highly energetic radical intermediate [AB+°*] tends to undergo fragmentation (or rearrangement) as a stabilizing process, this is responsible for the lower mass fragment ions present in the spectra. 4 is a very low abundance process, but theoretically it can occur. • 5 is a process which can occur at higher pressures (self Chemical Ionization), this is especially problematic in the ionization of alcohols and amines where you may find that the dominant ionization process is proton exchange between two analyte molecules, leading to the formation of the [M+H]+ pseudo-molecular ion.

22. EI M + e– M+· + e – + e – 70 eV Molecular ion ~ 55 eV 0.1 eV Ionization energies of valence electrons in formaldehyde. Here are other first ionization energies: CH3CH2CH2CH3 10.6 eV (sigma) CH2=CHCH2CH3 9.6 eV (pi) (CH3CH2)2O 9.6 eV (nonbonding) C5H10NH 8.6 eV (nonbonding) C6H6 9.2 eV (pi)

23. Electron ionization (70 eV) mass spectra of two isomeric ketones with the composition C6H12O.

24. The Molecular Ion Peak In the example above, involving benzamide (C7H7NO), the molecular ion (M+) peak had a mass to charge ratio (m/e) of 121. This value is calculated using the most abundant isotopes of the elements present in the molecule:7 × 12C = 84 7 ×1H = 7 1 ×14N = 14 1 ×16O = 16 Total 121 This type of calculation is called the unit mass molecular ion. Please refer to the spectrum for benzamide. Mass spectrum for benzamide.

25. The observed abundance of the suspected molecular ion peak must correspond to expectations based on the assumed molecular structure. For example, highly branched substances undergo fragmentation readily; therefore, these types of substances will not have a very intense molecular ion peak. The intensity of molecular ion peaks will usually correspond to the following sequence: aromatic compounds > conjugated alkenes > alicyclic compounds > sulfides > unbranched hydrocarbons > mercaptans > ketones > amines > esters > ethers > carboxylic acids > branched hydrocarbons > alcohols For instance, if an unknown compound is suspected to be an alcohol, then the molecular ion peak will be quite small or completely absent.

26. Interpreting spectra A simple spectrum, that of methanol, is shown here.   CH3OH+. (the molecular ion) and fragment ions appear in this spectrum.  Major peaks are shown in the table next to the spectrum.   The x-axis of this bar graph is the increasing m/z ratio.  The y-axis is the relative abundance of each ion, which is related to the number of times an ion of that m/z ratio strikes the detector.  Assignment of relative abundance begins by assigning the most abundant ion a relative abundance of 100% (CH2OH+ in this spectrum).  All other ions are shown as a percentage of that most abundant ion.  For example, there is approximately 64% of the ion CHO+ compared with the ion CH2OH+ in this spectrum.  The y-axis may also be shown as abundance (not relative).  Relative abundance is a way to directly compare spectra produced at different times or using different instruments. EI ionization introduces a great deal of energy into molecules.  It is known as a "hard" ionization method.  This is very good for producing fragments which generate information about the structure of the compound, but quite often the molecular ion does not appear or is a smaller peak in the spectrum. Of course, real analyses are performed on compounds far more complicated than methanol.  Spectra interpretation can become complicated as initial fragments undergo further fragmentation, and as rearrangements occur.  However, a wealth of information is contained in a mass spectrum and much can be determined using basic organic chemistry "common sense".

28. Following is some general information which will aid EI mass spectra interpretation: Molecular ion (M .+):If the molecular ion appears, it will be the highest mass in an EI spectrum (except for isotope peaks discussed below).  This peak will represent the molecular weight of the compound.  Its appearance depends on the stability of the compound.  Double bonds, cyclic structures and aromatic rings stabilize the molecular ion and increase the probability of its appearance. Reference Spectra: Mass spectral patterns are reproducible.  The mass spectra of many compounds have been published and may be used to identify unknowns.  Instrument computers generally contain spectral libraries which can be searched for matches. Fragmentation:General rules of fragmentation exist and are helpful to predict or interpret the fragmentation pattern produced by a compound.  Functional groups and overall structure determine how some portions of molecules will resist fragmenting, while other portions will fragment easily.  A detailed discussion of those rules is beyond the scope of this introduction, and further information may be found in your organic textbook or in mass spectrometry reference books.  A few brief examples by functional group are described (see examples). Isotopes:Isotopes occur in compounds analyzed by mass spectrometry in the same abundances that they occur in nature.  A few of the isotopes commonly encountered in the analyses of organic compounds are below along with an example of how they can aid in peak identification.

29. http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/MassSpec/masspec1.htmhttp://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/MassSpec/masspec1.htm

30. Four possible fragmentation pathways for the molecular ion of 2-hexanone.

31. Ions with no nitrogenor an even # N atoms odd-electron ionseven-number mass even-electron ionsodd-number mass Ions having anodd # N atoms odd-electron ionsodd-number mass even-electron ionseven-number mass Mass Spectrometry The Nitrogen Rule If a compound contains an even number of nitrogen atoms (or no nitrogen atoms), its molecular ion will appear at an even mass number. If, however, a compound contains an odd number of nitrogen atoms, then its molecular ion will appear at an odd mass value. This rule is very useful for determining the nitrogen content of an unknown compound. In the case of benzamide, the molecular ion appears at m/e = 121, indicating an odd number of nitrogen atoms in the structure. The masses of molecular and fragment ions also reflect the electron count, depending on the number of nitrogen atoms in the species.

32. 4-methyl-3-pentene-2-one has no nitrogen so the mass of the molecular ion (m/z = 98) is an even number N,N-diethylmethylamine has one nitrogen and its molecular mass (m/z = 87) is an odd number. A majority of the fragment ions have even-numbered masses (ions at m/z = 30, 42, 56 & 58 are not labeled), and are even-electron nitrogen cations.

33. Electron ionization (70 eV) mass spectra of molecular ion region of benzene (C6H6) and biphenyl (C12H10). Intensity of M+1 relative to molecular ion for CnHm : Intensity = n × 1.08% + m × 0.012% Contribution from 13CContribution from 2H

34. Methyl Bromide: An example of how isotopes can aid in peak identification. The ratio of peaks containing 79Br and its isotope 81Br (100/98) confirms the presence of bromine in the compound.

36. Element Isotope RelativeAbundance Isotope RelativeAbundance Isotope RelativeAbundance Carbon 12C 100 13C 1.11 Hydrogen 1H 100 2H .016 Nitrogen 14N 100 15N .38 Oxygen 16O 100 17O .04 18O .20 Sulfur 32S 100 33S .78 34S 4.40 Chlorine 35Cl 100  37Cl 32.5 Bromine 79Br 100 81Br 98.0 Relative Isotope Abundance of Common Elements:

37. Electron ionization mass spectrum (70 eV) of 1-bromobutane. Mass spectrum showing natural isotopes of Pb observed as an impurity of brass.

38. Molecular Mass and Nominal Mass The unit of atomic mass is the dalton, Da, defined as 1/12 of the mass of 12C. Mass specrometrists perfer the symbol “u” for “unified atomic mass unit”. “Da” and “u” are synonymous. Atomic mass is the weighted average of the masses of the isotopes of an element. Ex. Bromine 79Br 78.91834 Da 50.69% 81Br 80.91629 Da 49.31% Br = 78.91834 × 0.5069 + 80.91629 × 0.4931 = 79.904 Da The molecular Mass of a molecule or ion is the sum of atomic masses listed in a periodic table. Ex. C2H5Br = 12.0107 × 2 + 1.00794 × 5 + 79.904 × 1 = 108.965 The nominal mass of a molecule or ion is the the integer mass of the species with the most abundant isotope of each of the constituent atoms. Ex. C2H5Br = 12× 2 + 1× 5 + 79× 1 = 108

39. Chemical ionization (CI) CI uses a reagent ion to react with the analyte molecules to form ions by either a proton or hydride transfer: MH + C2H5+ MH2+ + C2H4 MH + C2H5+ M+ + C2H6 The reagent ions are produced by introducing a large excess of methane (relative to the analyte) into an electron impact (EI) ion source. Electron collisions produce CH4+ and CH3+ which further react with methane to form CH5+ and C2H5+: CH4+ + CH4 CH5+ + CH3 CH3+ + CH4 C2H5+ + H2

40. Chemical ionization uses ion-molecule reactions to produce ions from the analyte. The chemical ionization process begins when a reagent gas such as methane, isobutane, or ammonia is ionized by electron impact. A high reagent gas pressure (or long reaction time) results in ion-molecule reactions between the reagent gas ions and reagent gas neutrals. Some of the products of these ion-molecule reactions can react with the analyte molecules to produce analyte ions. Example (R = reagent, S = sample, e = electron, . = radical electron , H = hydrogen): R + e  R+.+ 2e R+. + RH  RH+ + R. RH+ + S  SH+ + R (of course, other reactions can occur)

41. CI Reagent Gases • Methane: • good for most organic compounds • usually produces [M+H]+, [M+CH3]+ and [M+C3H5]+ adducts • adducts are not always abundant • extensive fragmentation • Isobutane: • usually produces [M+H]+, [M+C4H9]+ adducts and some fragmentation • A dducts are relatively more abundant than for methane CI • not as universal as methane • Ammonia: • fragmentation virtually absent • polar compounds produce [M+NH4]+ adducts • basic compounds produce [M+H]+ adducts • non-polar and non-basic compounds are not ionized

42. a positive-ion CI reagent-gas mass spectrum of ammonia

43. Mass spectra of the sedative pentobarbital, using EI (left) or CI (right). The molecular ion ( m/z 226) is not evident with EI. The dominant ion from CI is MH+(protonated molecule). The peak at m/z 255 in the CI spectrum is from M(C2H5)+.

44. Fast Atom Bombardment (FAB)/Liquid Secondary Ionisation (LSIMS) This technique, developed in the early 1980s, revolutionised the range of compounds analysable by mass spectrometry and opened up the field to most areas of biomedical research. Although now considered insensitive by comparison with more recently introduced ionisation modes, FAB still has a role as a rapid, reliable and robust technique for samples where quantity and purity are not a problem. The sample is first dissolved in a liquid matrix. This is typically a viscous, low vapour pressure liquid such as glycerol or 3-nitrobenzyl alcohol. A few micro-litres of this liquid are placed on a small metal target at the end of a probe which is inserted into the mass spectrometer. The liquid surface is then bombarded with a beam of high kinetic energy atoms (xenon or argon) or ions (caesium). Molecules are sputtered from the surface, enter the gas phase and ionise, either by protonation or deprotonation. The resulting ions tend to be stable and exhibit little fragmentation.

45. Theory The techniques of FAB and LSIMS involve the bombardment of a solid analyte + matrix mixture by a fast particle beam (see figure). The matrix is a small organic species (glycerol or 3-nitrobenzyl alcohol, 3-NBA) which is used to keep a 'fresh' homogeneous surface for bombardment, thus extending the spectral lifetime and enhancing sensitivity. In FAB, the particle beam is a neutral inert gas, typically Ar or Xe, at bombardment energies of 4-10 KeV, whereas in LSIMS, the particle beam is an ion, typically Cs+, at bombardment energies of 2-30KeV. The particle beam is incident at the analyte surface, where it transfers much of its energy to the surroundings, setting up momentary collisions and disruptions. Some species are ejected off the surface as positive and negative ions by this process, and these 'sputtered' or secondary ions are then extracted from the source and analysed by the mass spectrometer. The polarity of the source extraction can be switched depending on what species are to be analysed. In the figure, the extraction is negative and thus only the positive ions are being analysed, this is termed +FAB. For LSIMS the atom beam is replaced by an ion beam. Both FAB and LSIMS are comparatively soft ionization techniques, and are thus well suited to the analysis of low volatility species, typically producing large peaks for the pseudo-molecular ion species [M+H]+ and [M–H]-, along with structurally significant fragment ions and some higher mass cluster ions and dimers.

46. Schematic of a Fast Atom Bombardment source. The figure shows a schematic representation of a fast atom bombardment ion source, operating in the positive ion mode. The fast atom beam is incident at the analyte+matrix surface where it causes the 'sputtering' off of secondary ions. The positive ions are then extracted from the source into the mass spectrometer.

47. Fast Atom Bombardment (FAB). The Cesium ions bombard the matrix.

48. Matrix-assisted Laser Desorption/Ionization (MALDI) Unlike FAB/LSIMS, this process uses a crystalline, rather than liquid, matrix, and a beam of photons, rather than atoms or ions. The net result is a dramatic increase, compared with FAB, in both sensitivity and mass range of analysable compounds. The sample is dissolved in a matrix and is allowed to crystallise on a stainless steel target. Suitable matrices include 2, 5 -dihydroxybenzoic acid and sinapinic acid. The target is then inserted into the mass spectrometer and the surface bombarded with a pulsed laser beam (typically generated by inexpensive nitrogen lasers with a beam wavelength of 337nm). Molecules are desorbed from the surface and ionise, usually by protonation or deprotonation. Any fragment or multiply charged ions are generally of low abundance in this ionisation mode.

49. (i) The Formation of a 'Solid Solution'. The analyte molecules are distributed throughout the matrix so that they are completely isolated from one other. This is necessary if the matrix is to form a homogenous 'solid solution' (any liquid solvent(s) used in preparation of the solution are removed when the mixture is dried before analysis). (ii) Matrix Excitation. Some of the laser energy incident on the solid solution is absorbed by the matrix, causing rapid vibrational excitation, bringing about localized disintegration of the solid solution, forming clusters made up of a single analyte molecule surrounded by neutral and excited matrix molecules. The matrix molecules evaporate away from these clusters to leave the excited analyte molecule. (iii) Analyte Ionization. The analyte molecules can become ionized by simple protonation by the photo-excited matrix, leading to the formation of the typical [M+X]+ type species (where X= H, Li, Na, K, etc.). Some multiply charged species, dimers and trimers can also be formed. Negative ions are formed from reactions involving deprotonation of the analyte by the matrix to form [M-H]- and from interactions with photoelectrons to form the [M]-° radical molecular ions. These ionization reactions occur in the first tens of nanoseconds after irradiance, and within the initial desorbing matrix/analyte cloud. It is in this way that the characteristic MALDI spectra are created, typically giving large signals for species of the type [NM+X]n+ (N * n).

50. MALDI. Formation of ions by laser desorption http://masspec.scripps.edu/information/intro/chapter3.html#3.2.1.