Mass Spectrometry Hyphenated Techniques GC-MS LC-MS and MS-MS
Reasons for Using Chromatography with MS Mixture analysis by MS alone is difficult Fragmentation from ionization (EI or CI) Fragments from each component in one spectrum yields intractable interpretation Suppression effects in soft techniques (ESI) Analytes that are less abundant or ionize poorly may not be detected Quantitation of mixtures is less accurate and less sensitive than quantitation of components that have been chromatographically separated
Chromatography -MS Data Rather than averaging all data into one spectrum, each full scan is saved separately to create a chromatogram TIC - Total Ion Chromatogram EIC - Extracted Ion Chromatogram Scan speed is critical for resolution of close-eluting components peaks could be missed or averaged together spectral tilting Intensities of low/high mass ions skewed
Chromatography -MS Data Identification of Components Background subtraction can be useful in cleaning up spectra Can remove background ions Can remove ions from co-eluting tailing compound EIC can support/contradict that two MS peaks are from the same/different compounds Comparing whether time profiles overlap EIC can show if ions come from the peak or are in the background
Offline LCMS LC may also be coupled with MS by fraction collection (ion exchange, size exclusion chromatography, TLC, Flash Column) Fractions may be analyzed by any compatible MS technique Offline Automation LC eluent can be spotted onto a MALDI target using a robot for MS analysis
LC in line with MS Liquid chromatography may be directly coupled to mass spectrometry using an APCI or ESI source Typical flow rates: 100 µl/min-2.5ml/min: APCI source > 1 ml/min: ESI source, usually need splitter ( 4.6 mm column) 1 µl/min - 1 ml/min: ESI source (200 µm- 4.6 mm column) < 1 µl/min: nanospray source ( 150 µm column)
Types of LC coupled inline to MS Most common= reversed phase (i.e. C18, C8, C4) Ion exchange Normal phase Affinity 2D Chromatography (Ion Exchange followed by reversed phase)
Restrictions Non-volatile salts should be avoided as signal suppression will result Strong ion-pairing agents such as trifluoroacetate should similarly be avoided Need polar solvents for ESI Normal phase not compatible with ESI (Use APCI) Additives must be compatible with ionization mode (i.e. ph of separation must match desired ions) Or use post-column addition
GCMS With the advent of capillary columns, GC columns can be inserted directly into the ionization source GC is typically coupled to EI, CI, and FI GC-MS is applicable to a narrower range of compounds due to volatility requirements When GC/MS is applicable, it is a fairly trouble free technique
Tandem Mass Spectrometry (MS/MS or MS n ) Soft ionization techniques such as ESI only result in parent ions, revealing no structural information Hard ionization techniques yield fragments, but specific precursor-product relationships cannot be obtained Isolate and fragment precursor ion and measure m/z of product ions to obtain structural information One mass analyzer isolates, another scans for products
Common Methods of Fragmentation Threshold Dissociation (Weakest bonds are broken) Collision Induced Dissociation (CID) Apply an electric field to accelerate precursor ions and induce bond cleavage via collision with neutral gas molecules (Ar, N 2 ) Infrared Multiphoton Dissociation (IRMPD) Use infrared laser to break bonds Electron Capture Dissociation (ECD) (Random) Multiply-charged cations capture electrons, inducing oddelectron fragmentation
CID + Ar + + Accelerated Precursor Ion Fragmentation through one or more collisions + Product Ions (And Neutrals)
IRMPD + hν + + Isolated Precursor Ion Fragmentation by one or more photons + Product Ions (And Neutrals)
ECD/ETD + e - or M - + + Isolated Precursor Ion Capture of one or more electrons Product Ions (And Neutrals)
Instruments for MS/MS Instruments where one or more analyzers are placed in series Triple Quadrupole (CID at ev levels) Four Sector (CID at kev levels) Quadrupole-Time-of-Flight (Q-TOF) (CID ev) TOF-TOF Trapping Instruments Quadrupole Ion Trap (CID ev, IRMPD, ECD) FT-ICR (CID, SORI-CID, IRMPD, ECD)
Quadrupole Ion Trap M+H MS/MS is done in one analyzer - Precursor Ion is Isolated by selective ejection - Precursor ion is given kinetic energy - Fragments are formed and trapped - Trap is scanned to detect products - Products can be isolated and fragmented etc.
Tandem Mass Spectrometry in an Ion Trap 1) Measure Full Spectrum 2) Ion Isolation: e.g., Precursor m/z 426.7 ± 1.5 50 Relative Abundance100 426.7 700.2 852.3 1138.7 300 900 1500 2000 m/z 426.7 3) Add Collision Energy 4) Measure m/z of Fragment Ions [For Peptide SLNVALR] Fragmentation of 426.7 SLNVAL SLNVA SLNV SLN SL S R LR ALR VALR NVALR LNVALR Courtesy of the MSCLS at the Univ. of Minn.
Triple Quadrupole Mass Analyzer Sample Inlet Ion Guide Q1 Q2 (Collision cell) Q3 EM Detector
Product Ion Scan Mode in a Triple Quadrupole Q1 Mass selection Q2 collision cell Q3 Full Scan A B C Ion C C+Ar Products Products D Ions in Source Q1 only transmits Ion C Fragment Ion C Q3 Scans for products
Multiple Reaction Monitoring (MRM) in a Triple Quadrupole Q1 Mass selection Q2 collision cell Q3 Mass Selection A B D C Ion C C+Ar C1+C2+C3 Ion C2 Ions in Source Q1 only transmits Ion C Fragment Ion C Q3 only transmits Ion C2
Neutral Loss Scan Mode in a Triple Quadrupole Q1 Pass A-H 2 O Q2 collision cell Q3 Pass A Ions A-F Ion A-H 2 O Ion B Ion C Ion D A-H 2 O A + +H 2 O A Ion E Ion F Scans across mass range Fragment ions one at a time Scans across mass range at 18 amu lower than Q1
Precursor Ion Scan Mode in a Triple Quadrupole Q1 Mass selection Q2 collision cell Q3 pass only 79 Ions A-F Ion A-PO 4 Ion B Ion C Ion D PO 3-79 Ion E Ion F Scans across mass range Fragment ions one at a time Transmits only 79
Precursor ion of 79 (-) -/+ Polarity switch Enhanced resolution (+) -/+ Polarity switch Charge state determination MSMS (+)
Intensity (cps) 7.5e8 7.0e8 6.5e8 6.0e8 5.5e8 5.0e8 4.5e8 4.0e8 3.5e8 3.0e8 2.5e8 2.0e8 1.5e8 1.0e8 5.0e7 β-casein Digest Total Ion Chromatogram 0.0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 Courtesy of the MSCLS at the Univ. of Minn. Time, min
Negative Precursor Ion Chromatogram 1.29e8 1.20e8 1.10e8 1.00e8 Intensity (cps) 9.00e7 8.00e7 7.00e7 6.00e7 5.00e7 4.00e7 3.00e7 2.00e7 1.00e7 0.00 5 10 15 20 25 30 35 40 45 50 55 60 65 Time, min Courtesy of the MSCLS at the Univ. of Minn.
2100 1900 Precursor Ion Scan at 30 min 1029.2 1700 Intensity, cps 1500 1300 1100 900 700 500 300 100 400 500 600 700 800 900 1000 1100 1200 m/z, amu Courtesy of the MSCLS at the Univ. of Minn.
Intensity, cps 1.10e5 1031.3 1031.7 1.00e5 9.00e4 8.00e4 7.00e4 6.00e4 Positive Enhanced Resolution Scan +2 ion, MW= 2060.6 5.00e4 4.00e4 1032.2 3.00e4 2.00e4 1.00e4 1031 1032 1033 m/z, amu Courtesy of the MSCLS at the Univ. of Minn.
MSMS on ion 1031.3 for sequence determination 3.4e6 a1 I F Q ps E E Q Q Q T E D E 3.0e6 Intensity, cps 2.6e6 2.2e6 1.8e6 1.4e6 1.0e6 6.0e5 2.0e5 y2 b2 y3 y6 y1-nh3 b2-nh3 y3-nh3 y4 y5 b5 y1 a1-nh3 a2-nh3 b1-nh3y2-nh3 b3 b3-nh3 y5-nh3 b4-nh3 a5y6-nh3 y4-nh3 b4 y8 y10 y7 y9 y10-nh3 y7-nh3 y11 y9-nh3 y11-nh3 y12-nh3 b6 b7 b8 y8-nh3b8-nh3b9-nh3 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z, amu Courtesy of the MSCLS at the Univ. of Minn.
Q-TOF Mass Analyzer Sample Inlet MCP Detector Ion Guide Q1 Q2 (Collision cell) Hexapole TOF Pusher Reflectron Select 1 ion in Q1 (Product Scan) Scan Q1 (Precursor Scan) Cannot perform Neutral Loss Scan
FT-ICR-MS M+H MS/MS is done in one analyzer RF fields can selectively isolate one ion RF fields (on or off resonance) can accelerate ions followed by collisions and detection of products IR laser or electron source can fragment isolated ions followed by detection of products Processes can be mixed, matched, repeated etc.
MS 4 With FT-ICR CO 2 - CO 2 - - O 2 C Reed et. al. JACS, 122, 10689-10696, (2000)
MS 6 With FT-ICR - O 2 C CO 2 - CID CO 2 - CID - - O t-bush - O - SO 2 - low energy O - H high energy CID t-bush No Reaction Phenol Proton Transfer Reed et. al. JACS, 122, 10689-10696, (2000)
Multiple ions at m/z 92
MS/MS Applications Structural elucidation aid for unknown compounds Functional group confirmation Positive Identification of known compounds environmental contaminants biological metabolites controlled substances (illegal drugs, steroids etc.) When Combined with Chromatography Screening of mixtures for specific moieties Structure-specific quantitation Thermochemical information (BDE etc.) Chiral analysis