Ch 13. Basics of Mass Spectrometry (I) : Principles & Ionization Sources Why should you be interested in mass spectrometry (MS)? - to identify unknown compounds - to quantify known materials - to elucidate the structural and chemical properties of molecules - detection of compounds at 10-12 g, 10-15 mole for a compound of mass 100 dalton 1 1. Where are MS used? Biotechnology: analysis of proteins, peptides, oligonucleotides Pharmaceutical Analysis: drugs discovery, combinatorial chemistry, pharmokinetics, drug metabolism Clinical Examination: neonatal screening, haemoglobin analysis, drug testing Environmental Analysis : water, food, air quality (PCBs etc) Geological Analysis : oil composition 2
1.1 Instrumental Design of Mass Spectrometer Ionization/ desorption Source Form ions (charged molecules) Mass Sorting Analyzer Sort Ions by Weight (m/z) Detection Ion Detection Detect ions Inlet Solid Liquid Vapor Sample Introduction Method to vaporize sample 100 75 50 25 0 1330 1340 1350 Mass Spectrum Data Analysis 3 Turbo pumps Diffusion Pumps Rough pumps Rotary pumps High Vacuum System Inlet Ion source Mass Filter Detector Data System HPLC Samle plate GC Solids probe DCI Electrospray MALDI FAB LSIMS EI CI TOF Quadrupole Ion Trap Magnetic Sector FT-ICR Microchannel Plate (MCP) Electron Multiplier 4
1.2 Vacuum Technology Pressure Gauges Gauge Pressure Range Typical Use (torr) Manometer 760-1 systems near atm Thermocouple gauge 1-10 -3 monitoring mechanical pumps Ionization gauge 10-3 -10-11 high-vacuum systems Vacuum Pumps Pump Lowest Attainable Pressure Typical Use Mechanical pump 10-2 -10-3 roughing or backing pump Diffusion pump 10-6 vacuum lines Turbomolecular pump 10-9 high-vacuum systems Cryopump < 10-10 ultrahigh-vacuum sys. 5 1.3 Reason for Vacuum in MS 10-6 to mid 10-5 Torr Increase sensitivity Avoid ion-molecule reactions Collision free ion trajectories Increase filament lifetime Avoid electric discharge Avoid background interference 6
2. Sample Introduction Techniques Direct Probe/Metal Targets Sample put onto the end of long probe and inserted into the MS Sample spotted with matrix onto a metal plate Gas chromatography: EI, CI, NCI - Sample must be volatile, thermally stable Liquid chromatography: PB, FAB, APCI, ESI Widely used in pharmaceutical industry LC/MS applicable to thermally labile, high MW compounds LC/MS suitable for proteins & peptides On-line technique 7 3. Ionization Methods Ionization Techniques Electron Impact (EI) Chemical Ionization (CI) Fast Atom Bombardment (FAB) ESI MALDI Hard Ionization Fragments Soft Ionization Intact 8
3.1 Electron Ionization (EI) M e - (70 ev) M (5 ev) 2e - (65 ev) Excess E (65eV) in the molecule leads to some degree of fragmentation M : molecular ions fragment ions neutral fragments M e - : M - (100 times less efficient) Energetic process. A heated filament emits electrons which are accelerated by a potential difference of usually 70eV into the sample chamber. Ionization of the sample occurs by removal of an electron from the molecule thus generating a positively charged ion with one unpaired electron. 9 1) Schematics of EI N M e - M. 2e - Fragmentation M. A B M. A B 10
2) Characteristics of EI Molecular ion AB e AB 2e Fragment ion AB e A B 2e Standard ionization methods in MS Vaporized sample bombards with high energy electrons (~ 70 ev) Hard ionization method leads to significant fragmentation Ionization is efficient but non-selective Widely used technique when coupled to GC Suitable for volatile organic compounds eg. hydrocarbons, oils, flavors, fragrances Not really coupled to LC today Produces M. radical cation giving molecular weight Produces abundant fragment ions Library searchable spectra 11 Electron Energy Profiles of Ionization Ionization efficiency 70 V 0 25 50 80 100 Electron energy (ev) 70eV : strong enough to knock off an electron and initiate fragmentation. 12
EI Spectra 13 14
3.2 Chemical Ionization (CI) Positive Ion Chemical Ionization (PCI or CI) Negative Ion Chemical Ionization (NCI,NICI or ECNI) 1. Instrumentation for CI 2. Selection of Reagent Gas 3. Analytical Application 15 1) EI CI Comparison Source pressure Mean free path EI 10-6 torr -200 mm CI 0.2 2 torr -2 x 10-4 torr High Energy process Odd E - ions formed Low Energy process (possess thermal energy) Even E - ions formed 16
2) Advantages and Disadvantages of CI Advantages of Chemical ionization: 1. Large (M1) ion identifies molecular weight (M) 2. Sensitivity is enhanced by - simple fragmentation (fewer peaks of higher abundance) - direct GC/MS interface 3. CI spectra complement EI spectra Disadvantages of Chemical ionization : 1. Simple fragmentation gives little structural information 2. Easy of ion source contamination 17 3) What happens in the CI source? Reactant ions are formed via EI/CI: CH 4 e - CH 4 2e - CH 4 e - CH 3 H 2e - CH 4 e - CH 2 H 2 2e - CH 4 CH 4 CH 5 CH 3 CH 3 CH 4 C 2 H 5 H 2 CH 2 2 CH 4 C 3 H 5 2H 2 H At 1 torr, the major ions are those at 17amu (CH 5 ), 29amu (C 2 H 5 ), 41amu (C 3 H 5 ) 18
Sample Ionization: 1. Proton transfer: higher proton affinity than that of reagent CH 5 RH CH 4 RH 2 (MH) C 2 H 5 RH C 2 H 4 RH 2 (MH) 2. Alkyl addition: C 2 H 5 RH RH C 2 H 5 (M29) C 3 H 5 RH RH C 3 H 5 (M41) 3. Hydride abstraction : lower PA molecules (M-1) CH 5 RH CH 4 H 2 R C 2 H 5 RH C 2 H 6 R 19 CI Mass spectra of reagent gas? 20
3.3 Fast Atom Bombardment (FAB) Ion Source 8000V or 1KV Cesium ion beam MH [ or MX ] Ion desorbed from matrix Matrix Comatrix Proton X : Na, K, Li Sample and Matrix Advantage Soft Ionization(MH, MNa... ) : little fragmentation Easy adaptation of HRMS : accurate mass measurement Matrix can be useful as ref. ion 21 1) Schematics of FAB Ion Source FAB gun 6~10 kev Primary atom or ion beam Probe Secondary ions Through Vacuum Lock Sample/Matrix To mass analyzer Xe Xe Xe Xe 0 ionization acceleration neutralization Slow atoms Slow ions Fast ions Fast atoms 22
2) Mechanism of ion formation Impact of a high-energy atom or ion formation of a high-temperature, high density gas in the cavity that is formed at the point of impact Generation of additional ions secondary electrons, radicals, and exited neutral species Mass Spectrom. Rev., 5, 191 (1986) 23 3) Basic setup of FAB MS Xe/Ar Xe/Ar ions ions are are generated generated by by Electron Electron Impact(EI) Impact(EI) Accelerating(3-8 Accelerating(3-8 kev) kev) and and Focusing Focusing Neutralization Neutralization by charge exchange by charge exchange with with neutral neutral gas gas in the collision cell in the collision cell (( X (fast) X (fast) X X X(fast) X(fast) X ) X ) or by Neutralizing metal such or by Neutralizing metal such as as Al Al Fast Fast atom atom beam beam hits hits the the drop drop of sample on the target of sample on the target Emerging Emerging secondary secondary ions ions Mass Mass analysis analysis 24
4) Matrix of FAB Ionization Successful ionization by FAB is deeply dependent on the matrix selection for the analysis. Matrix requirements - Dissolve the sample to be analyzed, (usually 1 mg/100 ml) - Facilitate in the ionization of the sample - low volatility - Not undergo a chemical reaction with the sample. - Constantly replenish the surface with new sample - Minimize sample damage from the high-energy particle beam - Reduce damage to the sample by absorbing the impact of the primary beam - Prolongs the sample ion current by constantly replenishing the upper layer with the fresh sample - Reduces the binding energy of the sample molecules Functions - proton donor(ve mode) - proton acceptor(-ve mode) - solvent - sample reservoir - reagent 25 Chemical Structures of Matrix 26
Matrix Selection of FAB MS Mol. Formula M/Z Glycerol C3H8O3 92.0473 Best choice for polar compounds 1-thioglycerol C3H8O2S 108.0245 More volatile than glycerol, evaporates quickly 3-nitrobenzyl alcohol (NBA) C7H7NO3 153.0416 Best choice for less polar compounds and many organometallics 2-nitrophenyl octyl ether (NPOE) C14H21NO3 251.1521 Triethanolamine C6H15NO3 149.1052 magic bullet C4H10S2O2 154.0122 Matrix C24H38O4 dioctyl phthalate (DOP) [bis(2-ethylhexyl) phthalate] Characteristics Only FAB matrix with no reactive hydrogen Good matrix for negative-ion FAB Enhances [M-H]- formation Widespread contaminant in solvents, gives characteristic 149 peak in EI mass spectra Use care to avoid contaminating the mass spectrometer 390.54 384 sulfuric acid H2SO4 Good for some inorganics and organometallics phthalocyanine) Corrosive, use care 97.967 (e.g. copper 27 FAB MS Spectra by positive and negative ion modes Positive mode Negative mode M H [MH] M [M-H] H M Na [M Na] M Cl- [M Cl]- J. Agric. Food Chem.; 47(2); 588, (1999) 28
29 Applications of FAB MS Synthetic Peptides Recombinant DNA Proteins and Glycoproteins Carbohydrates Drug Metabolites (including intact congugates) Detergents (anionic, cationic, non-ionic, amphoteric) Biocides Petrochemicals Oil Additives Oil Field Chemicals Phospholipids 30
3.4 Matrix Assisted Laser Desorption Ionization (MALDI) Laser MH [ or MX ] Matrix Comatrix Proton X : Na, K, Li Sample & matrix Advantage High Sensitivity Soft Ionization(MH, MNa... ) Mixture Analysis Low Cost No Contamination 31 Selection of Matrix in MALDI Sinapinic Acid a-cyano-4-hydroxycinnamic acid 2,5 Dihydroxybenzoic acid Super DHB 3-Hydroxypicolinic acid HABA Proteins >10kDa Peptides<10kDa Neutral Carbohydrates, Synthetic Polymers Proteins, Glycosylated proteins Oligonucleotides Proteins, Oligosaccharides 32
Advantage High sensitivity, mass range Simple structure Easy operation & maintenance, low cost Fast acquisition and automation Disadvantage Accuracy, resolution [~10,000] delayed extraction, reflectron Different calibration for different mass range & experimental condition (laser, matrix) 33 MALDI/TOF 1. Sample is mixed with matrix & dried on target 4. Ions are accelerated by an electrical field to the same kinetic energy, and they drift (or fly) down a field free flight tube where they are separated in space. 20-30 kv Flight tube 5. Ions strike the detector at different times, depending on the mass to charge ratio of the ion. 2. Target is introduced into high vacuum of MS High vacuum High voltage 3. Sample spot is irradiated with laser, desorbing ions into the gas phase and starting the clock measuring the time of flight. Pulsed laser Tim e 6. A data system controls all instrument parameters, acquires the signal vs. time, and permits data processing. 34
Higher resolution Higher mass accuracy PSD(Post Source Decay) Reflector for TOF Second Detector Reflector (Ion mirror) First Detector Ion Gate Laser The reflector increases the overall path length for an ion and it corrects for minor variation in the energy spread of ions of the same mass. Both effects improve resolution. 35 Applications of MALDI/TOF-MS Molecular biology, biochemistry Biopolymer Characterization Protein & peptides [proteomics] Sequence Analysis DNA, RNA [genomics] In- Source Decay Exonuclease Sequencing Carbohydrate Lipid Pharmaceutical Industry Combinatorial Chemistry Chemical Industry Synthetic Polymer Characterization 36
3.5 Atmospheric Pressure Chemical Ionization (APCI) Pressure = 760 torr Sample Inlet Nozzle (Lower Voltage) N 2 Solvent Flow From HPLC MH MH MH MH MH Heat Heat applied to metal sheath Corona discharge needle 37 APCI Simple and efficient device for the LC/MS analysis of compounds Needle : 3-6kV potential creates a corona discharge Ionization : by CI Soft ionization which generates pseudo molecular ions MH, (MNH 4 ) etc. (M-H) -, (MCH 3 COO) -, (MCl) - etc Generates singly charged ions, in general Suitable for less polar compounds compared to ESI Same degradation may occur in case of labile compounds 38
Characteristics of APCI Both positive and negative ions can preferentially be formed in the APCI source using a corona discharge Ion molecule reaction Positive ion mode - the creation of reactant positive ions in ambient air (the proton hydrates, H 3 O [H 2 O] n ) - the major primary ions N 2, O 2, H 2 O and NO are formed by electron impact of corona-created electrons on the major neutral components in air - Proton transfer reaction H 3 O [H 2 O] n T TH (H 2 O) m (n-m1)h 2 O if the gas basicity (proton affinity) of T is greater than that of water TH (H 2 O) m TH by stripped off in the gas curtain and declustering lens region 39 In negative ion mode, - electron created by the corona are rapidly thermalized (lose excess energy) through collision with neutrals and captured by electronegative species such as O 2 to form O 2- and O -. - super oxide (O 2- ) and its hydrates (O 2- [H 2 O] n ) and cluster (O 2- [O 2 ] n ) Ion Molecule Reaction Charge transfer R T T R R - T T - R Proton transfer RH T TH R R - TH T - RH 40
APCI MS spectra of Phenylbutazones 41 APCI MS/MS spectra 42
3.6 Electrospray Ionization (ESI) 43 1) Characteristics of ESI Soft ionization, which generates pseudo molecular ions MH, (MNH 4 ), (MNa), (MK) etc. (M-H) -, (MCH 3 COO) -, (MCl) - etc. Applicable for wide range of compounds with relatively high sensitivity Up to 150 kda in case of proteins Middle to High polar compounds Thermally labile compounds Non covalently binding complex Generates multiply charged ions of biopolymers (MnH) n / (M-nH) n- 44
ESI Mass Spectra 1picomole Myoglobin MW: 16,939.4 amu LC/MS analysis m/z 1230 Heme dimer Second series protein Heme Synthetic DNA MW: 4260.7 amu Negative ion mode 45 Useful mass differences for interpretation of ESI-MS - Positive ion mode - (MH) (MNH 4 ) 17 (MNa) 22 5 (MK) 16 (MMeCNH) 38 41 46