L17 - Lecture 17 High Res Mass Spec Hei et al....

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Unformatted text preview: Lecture 17 High Res Mass Spec Hei et al. (2001) Anal. Chem. 73:647-650. Intro to High Resolu0on MS •  High resolution – mass spectral peaks resolved to multiple decimal places •  Able to propose molecular formulas, helps with unknown identification •  Exact mass – every atom has a well defined atomic mass to many decimal places (includes binding energy) •  Nominal mass – atomic mass rounded to the nearest integer (low resolution MS) Exact (IUPAC) masses Element Symbol Hydrogen H Carbon C Nitrogen N 99.985 1.007825 0.015 2.014102 98.90 12.000000 1.10 13.003355 99.63 14.003074 0.37 15.000109 15.994915 0.04 16.999131 0.20 O Isotopic mass 99.76 Oxygen Abundance (%) 17.888160 Fluorine F 100 18.998403 Phosphorus P 100 30.973762 95.03 31.972072 0.75 32.971459 4.22 33.967868 75.77 34.968853 24.33 36.965903 50.69 78.918336 49.31 80.916289 Sulfur S Chlorine Cl Bromine Br Resolving Power Characteris5c Quadrupole Magne5c Sector Time of Flight Mass Range (Da) < 4000 15,000 “Unlimited” Resolving power 4000 102- 105 ~104 Resolving power is the ability to separate two neighboring peaks. This is a measure of how ‘sharpness’ of a mass peak. RP = m Δm Although quadrupoles can have RP = 4000, in reality they are only capable of unit resolu0on (nominal masses) for rou0ne work The Need for High Resolu0on Compound Nominal Mass Exact Mass 6:2 FTOH- Sulfate - C8H4SO4F13 443 442.96225 6:2 monoPAP F Molecular Formula - C8H5PO4F13 443 442.97177 FF FF F F O F F FF FF F 6:2 FTOH- Sulfate O FF FF F O S O F O- F FF FF F 6:2 monoPAP What are some other ways to dis0nguish between the two? -O O P OH Mass Spectrometer •  3 basic components to any mass spec (S A D) SOURCE ANALYZER •  Source produces ions from neutral analytes •  Analyzer separates ions of different masses •  Detector gives a signal when an ion strikes it D E T E C T O R Analyzer Review •  The analyzer used determines the resolu2on •  Quadrupole – acts as “mass filter” and has three basic modes 1) Pass all ions (RF mode) 2) Scan all ions 3) Selected ion monitoring •  Pros: High sensi0vity (esp. MS/MS in MRM mode), inexpensive, small size, fast scan •  Cons: Low resolu0on (unit masses only) Magne0c Sector •  Oldest mass spec design, though not always a high resolu0on instrument •  Basic opera0ng principles: –  Like quads, unwanted ions are filtered/destroyed and never reach detector –  Ions imparted with equal kine0c energies by source –  Flight path of ions through analyzer altered by a magne0c field (which selects by m/z) and electrosta0c field (which selects by kine0c energy) –  Ions with the correct combo of m/z and KE reach detector, all others lost Magne0c Sector - Physics •  All ions in source imparted with (hopefully) the exact same Kine0c Energy: q Vs = mv2 / 2 •  Force exerted on an ion by an external magne0c field (B) equals centripetal force: qvB = mv2 / r mv/q = Br But we don’t know v! Instead, sub in KE = mv2 / 2 Rearrange to get m/q = B2r2 / 2Vs •  Assuming all ions imparted with the same Vs then m/q will depend on the correct radius with variable B Magne0c Sector – Old Design •  Red ion has the correct m/z for the B that the magnet is currently set to •  Red ion given the radius of curvature needed to reach detector safely Magne0c Sector – Old Design •  Blue ion has the correct m/z for the B that the magnet is currently set to •  Blue ion not massive enough, path deflected too much by magnet, lost on walls •  In full scan mode, B is varied constantly with 0me Magne0c Sector – Old Design •  Main assump0on: all ions imparted with the same KE by the source (Vs is in our equa0on) •  Problem: Various factors combine to increase variability in KE •  Ions with same m/q but different KE will not be focused together, resul0ng in peak broadening Desired KE from source KE not quite right •  High resolu2on can not be obtained with a magne2c sector alone Magne0c Sector – New Design How do we overcome the kine2c energy problem? •  Remove undesired kine0c energies to sharpen mass spec peaks •  Use electrosta0c sector before/aker magne0c sector to destroy ions with the wrong KE •  Curved electric plates give ions with same KE the same radius of curvature •  Ions with different m/z unaffected by electrosta0c analyzer + - Electrosta0c Sector Electrosta0c Sector Physics •  All ions in source imparted with (hopefully) the exact same Kine0c Energy: q Vs = mv2 / 2 •  Force exerted by electrosta0c field (E) on a charged par0cle is balanced by centripetal force mv2 /r = qE •  But, KE = mv2 / 2 •  Hence r = 2Vs / E •  All q and m terms cancel out, leaving only dependence on kine0c energy! Electric/Magne0c Dual Sector •  Red ion has the correct m/z for the B that the magnet is currently set to •  Also has the ideal KE to pass through electrosta0c analyzer Electric/Magne0c Dual Sector •  Blue ion has the correct m/z for the B that the magnet is currently set to •  Does NOT have the ideal KE so destroyed by electrosta0c analyzer Electric/Magne0c Dual Sector •  Modern dual sector instruments capable of high resolu0on to 4 decimal places (some0mes 5) •  Exact mass determina0on possible – molecular formulas of unknowns can be proposed •  More advanced setups contain more than one electrosta0c analyzer (EBE design) •  Sector instruments need to be FAST •  Difficult to couple with LC (very strong vacuum needed) •  Scan/SIM analyses possible just like quads Time of Flight (ToF) Mass Spec •  •  Old and simple design, but not always high resolu0on Unlike quadrupole or sector, all ions “survive” passage from source to detector (sensi0vity boost) Always in “scan” mode, no SIM •  Basic Opera0ng Principles: •  –  –  –  –  Ions imparted with an exact kine0c energy from the source Ions with different masses have different veloci0es Ions separated in a flight tube according to their masses Transit 0me from source to detector determines mass Linear ToF (Old Design) Time of Flight Analyzer Source Detector From GC/LC m/z Low resolu5on! Why?? Linear ToF (Old Design) •  Need a star0ng “gun” to start the 0mer, all ions must be accelerated together •  Problem: Constant flow from GC/LC column, need a way to accelerate ions together at a specific 0me •  Solu0on: Pulse ions into ToF at right angles to column (orthogonal) •  Side effect: Ions travelling between pulses are lost (reduced sensi0vity) •  MALDI and ToF go well together Orthogonal Pulsing R E P E L L E R Source Accelera5on plates Time of Flight Analyzer Linear ToF (Old Design) •  Low resolu0on – mostly due to ions not having the exact same kine0c energy •  Can increase resolu0on by using a reflectron –  Uses an electrosta0c field to compensate for KE differences –  Lengthens the ion flight path Linear ToF (Old Design) Time of Flight Analyzer Source Detector From GC/LC Desired KE from source KE not quite right m/z Reflectron ToF (High Resolu0on) ++ +++ + m/z High resolu5on now possible Reflectron Detector Reflectron ToF •  Reflectron ToF allows high resolu0on •  Accuracy to four decimal places allows molecular formulas to be proposed •  Overall sensi0vity? –  No ions discarded while scanning, all reach detector –  Pulsing means many ions are never sent into the analyzer •  ToF can be interfaced with a GC source or LC source, requires orthogonal pulsing Quadrupole ToF •  Q- ToF is a tandem MS/MS technique •  Collision cell between quadrupole and 0me of flight analyzers •  Allows a parent ion to be chosen in the quadrupole which is then fragmented •  All daughters detected using the ToF •  Great sensi0vity and allows confirma0on of molecular structure Reflectron ToF Analyzer Ion Source Quadrupole Collision Cell Detector Pulser Reflectron ToF Analyzer Ion Source Quadrupole Collision Cell Detector Pulser ...
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This note was uploaded on 01/05/2012 for the course CHM 410 taught by Professor - during the Fall '11 term at University of Toronto- Toronto.

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