Russell Rouseff FOS 6355 Summer 2005

What is Mass Spectroscopy Analytical Chemistry Technique Used to identify and quantify unknown compounds Can also elucidate structure and chemical properties Sensitive technique As little as g can be analyzed Identification based on molecular fragmentation patterns fragments are separated by their m/z ratio

Early Mass Spectrometry Double focusing analyzer Time of Flight MS Preparative Mass Spectrometry Quadrupole MS GC/MS Identifying Organic Compounds Chemical Ionization Peptide Sequencing Atmospheric Pressure Ionization GC-C-IRMS Inductively Coupled Argon Plasma Protein Structure Protein Mass Mapping/Fingerprinting MS of a virus First proton transfer MS MS Time lines

Mass Spectroscopy Applications Detect and identify the use of steroids in athletes Monitor the breath of patients by anesthesiologists during surgery Determine the composition of molecular species found in space Determine whether honey is adulterated with corn syrup Monitor fermentation processes for the biotechnology industry Detect dioxins in contaminated fish Establish the elemental composition of semiconductor materials Perform forensic analysis – arson identification Determine exact atomic mass and isotope abundance

Objectives How did mass spectrometry originate? What is a mass spectrometer? What are the characteristics of a mass spectrum? How is the sample introduced into the mass spectrometer? How are the molecules and fragment ions produced in the ion source? How does the analyzer work? How does the detector work? What are the roles of computers in mass spectrometry?

Objectives How can mass spectrometric data be used for structure analysis? What other techniques are used to produce ions? How large a molecule can be analyzed? How is mass spectrometry used with GC,LC, and other separation techniques? How is mass spectrometry used for quantitative analysis? What is mass spectrometry/mass spectrometry (MS/MS)? What is isotope ratio mass spectrometry?

sourceanalyzer ion detection data system Vacuum pumps Sample Introduction Ion FormationIon SortingIon Detection Data Handling Data Output Mass spectrum

Sample Introduction Neutral molecules in gaseous state Can be either liquid, solid or gas under STP must volatilize under vacuum must be pure often coupled with GC, HPLC or CE but can use solids probe if pure

Ionization Techniques Electron Impact, EI Chemical Ionization, CI Fast Atom Bombardment, FAB Atmospheric Pressure Chemical Ionization, APCI Proton Transfer Reaction

Sample entrance filament Electron collector Ion focusing lenses repeller Ion Formation (EI) + + +

CO 2 + Molecular ion Base ion CO + O+O+ C+C+

Contains C, H, O

Sample Ionization Electron Impact, EI, most common 70 eV standard ionization energy M + e - => M + + 2e - But sometime 70 eV too powerful

EI may overfragment

Chemical Ionization A softer ionization technique Use ionized molecules to transfer protons (+) M + CH 5 + => (M+H) + + CH 4 Chemical Ionization gases methane isobutane

Chemical Ionization Produces spectra with little fragmentation However, no standard spectral libraries Spectra tends to be instrument specific reagent gas pressures, ionization efficiencies Must generate own standards

Large Molecule Ionization Can routinely analyze molecules of 10,000 Da or more Upper limit used to be 2,000 Da

APCI A form of chemical ionization Used for LC/MS interfaces Allows for removal of solvent before vacuum

Proton Transfer Mass Spectrometery Europhysics News (2004) Vol. 35 No. 6 glow discharge H30+H30+ H M => (M + H) = H 2 O H30+H30+ M+M+ M M+M+

Europhysics News (2004) Vol. 35 No. 6 Proton Transfer Mass Spectrometery Detects aroma compounds at ppb levels Produces spectra with little ionization, quantify using M +

Mass Analyzer Types Magnetic Sector Quadrupole mass analyzers Ion trap Time of flight

H0H0 Magnetic Field Magnetic Sector Mass Spectrometers Exit slit Molecular Source Ionization chamber e-e- detector

Magnetic Sector Disadvantages Not well-suited for pulsed ionization methods Very High cost Large size High maintence costs

resonant ion nonresonant ion detector source focusing lens quadrupole rods quadrupole mass analyzer Mass filters

Quadrupole Advantages Classical mass spectra Good reproducibility Relatively small/ compact Relatively low-cost systems Can improve sensitivity 100x with SIM

Quadrupole limitations Limited resolution Peak heights variable as a function of mass (mass discrimination). Peak height vs. mass response must be 'tuned'. Not well suited for pulsed ionization methods

Ion Trap Advantages High sensitivity Multi-stage mass spectrometry, MS n Compact mass analyzer

Ion Trap Limitations Requires careful quantitation Limited dynamic range Subject to space charge effects and ion molecule reactions No sensitivity gain using SIM

Time of Flight MS

MS/MS

ASMS- American Society for Mass Spectrometry Excellent educational MS resource with many MS related links. Cambridge University WWW Mass Spectrometry Server Cambridge University WWW Mass Spectrometry Server Comprehensive site with information on ionization processes, mass analyzers, and other mass spectrometry techniques. Organic Chemistry Online Introductory information about analysis of organic compounds by electron impact mass spectrometry, tables of common fragment losses. University of Leeds: Introduction to Mass Spectrometry University of Leeds: Introduction to Mass Spectrometry Introductory site focusing on the analysis of biochemical samples. Information on Specific Techniques General Mass Spectrometry Sites

Sheffield ChemPuter: Isotope Patterns Calculator Site predicts isotope pattern based on molecular formula. UCSF Protein Prospector Site includes useful programs for analyzing the mass spectra of proteins and peptides. Proteomics tools for mining sequence databases in conjunction with Mass Spectrometry experiments Misc. Mass Spectrometry Sites