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Unformatted text preview: Accurate Mass Spectrometry Lab Spectrometry
February 1, 2008 Jon Karty What is Resolution? Resolution is the ability to separate ions of nearly equal Resolution mass/charge mass/charge e.g. C6H5Cl and C6H5OF @ 112 m/z
12 C6H5Cl = 112.00798 amu (all 12C, 35Cl, 1H) Cl C, 35Cl, 12 C6H5OF = 112.03244 amu (all 12C, 16O, 1H, 19F) OF C, 16O, H, 19 Resolving power of 4600 required to resolve these two Two definitions Two Resolution = Δm / m (0.015 / 112.03 = 0.00013 or 1.3*10-4) Resolution Resolving power = m / Δm (112.03 / 0.015 = 7,468 or 7.5*10+3) Resolving High resolution, high accuracy MS can replace elemental High analysis for chemical formula confirmation analysis MAT-95 is capable of 60,000 resolving power LCT is capable of 5,000 resolving power MALDI-TOF-TOF has RP of 10,000-15,000 FTMS instruments can have RP > 105 to 106 High resolution facilitates high precision measurements High resolution is considered RP ≥ 5,000 High New “-omics” from MS New Metabolomics Application Application of accurate mass spectrometry to identify small molecule metabolites in biological systems systems
Sertraline’s Sertraline’s activity activity Fluoxetine’s metabolite has same activity, but longer halflife Accurate mass spectrometry determined chemical Accurate formula of THG (designer steroid) formula Petroleomics Application Application metabolite has significantly reduced SSRI of high resolution, accurate mass spectrometry to the study of fossil fuels spectrometry Resolving Power Example Resolving
100 100 RP= 5,000
100 RP= 7,000 C6H5Cl C6H5OF 80 80 80 In te n sity ( %) In te n sity ( %) 60 60 In te n sity ( %) 60 40 40 40 20 20 20 0 11 1.95 11 2.00 Mass [amu] 112.05 11 2. 10 0 11 1.95 11 2.00 Mass [amu] 112.05 11 2. 10 0 11 1.95 11 2.00 Mass [amu] 112.05 11 2. 10 All resolving powers are FWHM Calibration Calibration Calibration Calibration equation determines relationship between observed signals and actual m/z ratios between External Calibration: mass spectrum of calibration External compounds is acquired in a different experiment than the analyte compound than Instrument Power drift can introduce errors supply voltages, temperature in the lab, etc. Internal Internal Calibration: mass spectrum of calibration compounds are recorded at the same time as analyte compound analyte Provides most accurate data Requirement to observe both calibrant and analyte ions Requirement in same experiment can be a significant challenge in LockSpray LockSpray on LCT is a unique solution to internal calibration problem calibration Mass Accuracy Mass Mass spectrometer accuracy often reported as a relative Mass value value ppm = parts per million (1 ppm = 0.0001%)
5 ppm @ m/z 300 = 300 * (5/106) = ±0.0015 Th ±0.0015 5 ppm @ m/z 3,000 = 3,000 * (5/106) = ±0.015 Th ppm 3,000 ±0.015 High resolving power facilitates precise mass High measurements measurements Mass accuracies for MSF instruments MAT-95: <5 ppm is standard precision (int. calib.) LCT: <50 ppm (ext. calib.), <5 ppm (int. calib.) Accurate mass measurement is defined is better than 5 Accurate ppm error ppm Accurate mass spectrometry facilitates determination of Accurate chemical formula directly from mass spectrum chemical Formula Matching Basics Formula Atomic Atomic 14N
12 weights are not integers (except 12C)
11 = 14.0031 Da; 11B = 11.0093 Da; 1H = 1.0078 Da 14.0031 11.0093 19 56 16O = 15.9949 Da; 19F = 18.9984 Da; 56Fe = 55.9349 Da 15.9949 18.9984 Difference Difference Related from integer mass is called “mass defect” or “fractional mass” defect”
to binding energy of the nucleus Sum Sum H, of the mass defects depends on composition composition
N increase mass defect Hydrogen-rich Hydrogen-rich molecules have high mass defects Heptadecane (C17H36)= 228.2812 O, Cl, F, Na decrease it Hydrogen deficient species have low mass defects Morphine, (C17H19NO3) = 285.1365 More Formula Matching More Accurate Accurate mass measurements narrow down the possible formulae for a particular molecular weight weight 301 301 321 321 4 compounds within 0.0016 Da (5 ppm) of 321.1000. Mass Mass entries (150 formulae) in NIST’02 @ nominal MW spectrum and user info complete the picture picture Isotope Isotope (e.g. (e.g. distributions indicate/eliminate elements info eliminates others Cl, Br, Cu) no F, Co, I in reaction) User-supplied User-supplied Suggested C6H28O2 formula has to make chemical sense is not reasonable nor is Cl3H2Co4 Isomers are not distinguished in this analysis ESI Source Diagram ESI
3 – 4 kV 760 torr 45 V 5V 1 torr 10-3 torr 10-6 torr Characteristics of ESI Ions Characteristics ESI is a thermal process (1 atm in source)
fragmentation due to ionization (cf EI) Little Little e.g. Solution-phase ESI ions are preserved in MS organometallic salts ions are generated by ion transfer often generates multitply charged ions (M+H)+, (M+Na)+, or (M-H)-, rarely M+• or M-• Ions are almost always EVEN electron ions ESI (M+2H)2+ or (M+10H)10+ Most ions are 500-1500 m/z even for LARGE polymers ESI ESI spectrum x-axis must be mass/charge (m/z or Th, not amu or Da) or ...
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This note was uploaded on 01/18/2012 for the course C 613 taught by Professor Davide.clemmer during the Spring '08 term at Indiana.
- Spring '08