Introduction to Mass Spectrometry Introduction to Mass Spectrometry Eddy Esmans May th EU-Meeting on Cobalamins and Mimics Antwerp - Belgium

I. Introduction II. Ionization methods 1. Electron impact 2. Chemical ionization and DCI, NICI 3. FAB, SIMS, LD and MALDI 4. Field desorption 5. Electrospray ionization III. Analyzers 1. Magnetic sector 2. Quadrupole – ion trap 3. Fourier transform 4. Time of flight IV. MS/MS-methods

The Components of a Mass Spectrometer Inlet system Ion Source Analyzer Ion Detector Computer Mass Spectrum m/z

I. Introduction II. Ionization methods 1. Electron impact 2. Chemical ionization and DCI, NIC 3. FAB, SIMS, LD and MALDI 4. Field desorption 5. Electrospray ionization III. Analysers 1. Magnetic sector 2. Quadrupole – ion trap 3. Fourier transform 4. Time of flight IV. MS/MS-methods

II. Ionization methods 1. Electron impact E 70 eV electron M +. F i +. kr Unimolecular type kr = E – E 0 E N-1 E = internal energy of e.g. M +. E 0 = activation energy of a particular fragmentation N = degrees of freedom = frequency factor IONIZATION EFFICIENCY : ca. 1/1000

QET : Quasi Equilibrium Theory a.A polyatomic molecule does not fragment immediately but during ionization period of sec it undergoes a few vibrations. fragmentation is a “relative slow” process. b. The energy transferred to M is not localised but is statistically spread over the molecule. c. If the event occurs than this energy is concentrated at one particular bond. This bond will break here. d. The probability of breaking a particular bond in not a function of abundance. e. Metastable ions are formed : ions with a life time of > seconds.

AB AB +. ” 0 ” 1 ” 2 ” 3 ” 4 ” 5 Interconversion ’ 5 AB +. ’ 0 ’ 1 ’ 2 ’ 3 ’ 4 -I p (theoretical) E-impact-ionisation occurs according to the Frank-Condon-principle (vibration is 100 times slower than ionisation)

I. Introduction II. Ionization methods 1. Electron impact 2. Chemical ionization and DCI, NICI 3. FAB, SIMS, LD and MALDI 4. Field desorption 5. Electrospray ionization III. Analysers 1. Magnetic sector 2. Quadrupole – ion trap 3. Fourier transform 4. Time of flight IV. MS/MS-methods

M(g) + reagent gas [MH] + benefit: producing molecular mass information proton affinity !!! proton affinity PA of M > proton affinity PA of the reacting species Classical reagent gasses: Methane: CH 5 + NH 3 : NH 4 + (NH NH 3 NH NH 2. ) Isobutane: C 4 H 9 + PS : if PA(M)  PA(reagent gas) [MH] + + ADDUCT FORMATION [M + NH 4 ] + [M + C 2 H 5 ] + if PA(M) < PA(reagent gas) only adducts bad sensitivity 2. Chemical ionization (CI) and DCI, NICI

Desorption chemical ionization

Negative Ion Chemical Ionization Principle : ion souce is filled with CH 4 and 70 eV electrons are slowed down to thermal energy. These electrons can be “captured” by molecules containing sulphur (cfr. Electron capture GC) formation of M °- -ions

I. Introduction II. Ionization methods 1. Electron impact 2. Chemical ionization and DCI, NICI 3. FAB, SIMS, LD and MALDI 4. Field desorption 5. Electrospray ionization III. Analysers 1. Magnetic sector 2. Quadrupole – ion trap 3. Fourier transform 4. Time of flight IV. MS/MS-methods

Fast atom bombardment (FAB) and Secundary Ion Mass Spectrometry (SIMS) Principle: Ions (Cs + ) Neutrals (Ar, Xe, …) IONS analysed Sample 1. FAB : Ar + e Ar + acceleration (5-15 KeV) Ar + + Ar Ar + Ar + fastslow + 8 KeV fast 2. SIMS : Cs + generated (35 KeV) 3. LSIMS : Sputtering yield (number of particles ejected/incident particle) Dependent on mass and velocity of impinging particle

Matrix properties 1. Good solubility 2. Vapour pressure must be sufficiently low to maintain vacuum conditions 3. Viscosity must allow diffusion of the analyte from the bulk to the surface 4. Polar : to solvate and separate preformed ion glycerol, 3-nitrobenzylalcohol, mixture of 1,4-dithiothreitol/1,4-dithioerythitol 5:1 (magic bullet)

Laser Desorption & Matrix Assisted Laser Desorption A few lasers: N2 –laser : 337 nm Nd-Yag laser : 354 & 266 nm E: 20mJ/cm2

I. Introduction II. Ionization methods 1. Electron impact 2. Chemical ionization and DCI, NICI 3. FAB, SIMS, LD and MALDI 4. Field desorption 5. Electrospray ionization III. Analysers 1. Magnetic sector 2. Quadrupole – ion trap 3. Fourier transform 4. Time of flight IV. MS/MS-methods

Field desorption

I. Introduction II. Ionization methods 1. Electron impact 2. Chemical ionization and DCI, NICI 3. FAB, SIMS, LD and MALDI 4. Field desorption 5. Electrospray ionization III. Analysers 1. Magnetic sector 2. Quadrupole – ion trap 3. Fourier transform 4. Time of flight IV. MS/MS-methods

Picofrit columns™ Picofrit columns™ - injection: 1  l - flow-rate: 500 nl/min - isocratic 20/80 NH 4 Ac (0.01 M) / MeOH - column: AQUASIL C18, 75  m x 4.9 cm (15cm 2cm), tip 5  m

I. Introduction II. Ionization methods 1. Electron impact 2. Chemical ionization and DCI, NICI 3. FAB, SIMS, LD and MALDI 4. Field desorption 5. Electrospray ionization III. Analyzers 1. Magnetic sector 2. Quadrupole – ion trap 3. Fourier transform 4. Time of flight IV. MS/MS-methods

I. Ion source : Ions get kinetic energy V  8 KV V = tension m = mass v = speed z = charge

II. Electrostatic sector : E = electrostatic field = Ions with the same E kin will travel with the same r and leave the electrostatic sector at the same point (This is independant of their mass !!!)

III. Magnetic sector :

I. Introduction II. Ionization methods 1. Electron impact 2. Chemical ionization and DCI, NIC 3. FAB, SIMS, LD and MALDI 4. Field desorption 5. Electrospray ionization III. Analyzers 1. Magnetic sector 2. Quadrupole – ion trap 3. Fourier transform 4. Time of flight IV. MS/MS-methods

a. Quadrupole filter Quadrupole field: E = E 0 ( x +  y +  z) Independent field in x,y,z-directions. Ions entering this field will undergo a force F = eE

Quadrupole field subjected to the restraites imposed by the Laplace-equations: Physical meaning : the Laplacean is a measure for the distorsion of the E-field

Hyperbolean !!!

Applied potential 1. Equation of motion of the ions entering this field mx = eE x ¨ mx¨ ¨ x ¨ y ¨ xz and yz motion of ions in plane mz = 0¨ Velocity in z-direction is cte but ions are accelerated in x and y-directions ! -

Stability diagram  0 = U + V.cos  t (  = 2  f) x ¨ y ¨ Matthieu-equations U = V V = V

Stability diagram scanning : changing U and V but keeping what if U = 0resolution = 0 Rf-quadrupole only will be able to pass m/z-values > certain m/z-value as long as V is in stability area.

I. Introduction II. Ionization methods 1. Electron impact 2. Chemical ionization and DCI, NIC 3. FAB, SIMS, LD and MALDI 4. Field desorption 5. Electrospray ionization III. Analyzers 1. Magnetic sector 2. Quadrupole – ion trap 3. Fourier transform 4. Time of flight IV. MS/MS-methods

Ion cyclotron resonance Fourier Transform MS Ion can be trapped in a H-field circular motion with frequency Relation between  and m/z-value each m/z-value will move with its typical frequency/radius The ion will have a stable trajectory when :

Simultaneously excite all ions by electromagnetic pulse (1µs). Depending on their m/z-values ions will absorb energy at their frequency and subsequently get hifgher trajectories close to the receive plates. All the frequencies detected in this time ellaps by the receive plates at the same time.

I. Introduction II. Ionization methods 1. Electron impact 2. Chemical ionization and DCI, NIC 3. FAB, SIMS, LD and MALDI 4. Field desorption 5. Electrospray ionization III. Analyzers 1. Magnetic sector 2. Quadrupole – ion trap 3. Fourier transform 4. Time of flight IV. MS/MS-methods

Time of Flight Source : ion Resolution > Mass range flight tube : L

Time of Flight Reflectron : corrects for energy dispension

Time an ion spends in reflectron 2 ions with mass M correct energy E energy E’ t, t’ = flight time in the field free region of TOF

Ions come in the reflectron : penetrate a distance x or x’ a>1 E’kin>Ekin t’ flight x x’ = a 2 x Conclusion : a t flight but x’ < x

Tandem Mass Spectrometry M P M F penetration depth:

Time in reflectron to penetrate a distance n vivi v0v0 x Total time in reflectron to cover a distance of 2x

I. Introduction II. Ionization methods 1. Electron impact 2. Chemical ionization and DCI, NIC 3. FAB, SIMS, LD and MALDI 4. Field desorption 5. Electrospray ionization III. Analyzers 1. Magnetic sector 2. Quadrupole – ion trap 3. Fourier transform 4. Time of flight IV. MS/MS-methods