Ch. 22 Mass Spectrometry (MS)

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1 Ch. Mass Spectroetry (MS).1 MS easures ass of atos, olecules, or fragents of olecules -1. What is MS? Gaseous ato fro condensed phase ionized Accelerated & separated By ass to charge ratio /z M=00: 1 ion /z=00 ion /z=100 ass spectru : detector response vs. /z Peak area ~ abundance of isotope norinal ass: usual ass -1. What is MS?. Magnetic Sector MS Uses agnetic field : gas entering & ionization at ion ode acceleration by E. field expelled into analyzer tube : high vacuu to reduce collision with background gas agnet deflects ion toward D. depending on B at electron ultiplier detector (10 5 /electron)

2 How ions of different asses are separated by B?.3 Under electrical field ass:, charge: ze acceleration voltage: V Under agnetic field ion ze experiences a agnetic force B deflects ion through a radius r zebr = zev Kinetic E = potential E 1 v = zev v = zevb v = zevb r zebr v = zev Centripetal force = agnetic force z eb r = V If r fixed, then adjust B or V (~3kV) -1. What is MS?.4 1. Electron Ionization (EI) e - fro hot filaent. Accelerated through 70V. M e - M e - e - for HCHO 70eV olecular ~55eV 0.1eV ion (1eV=96.5kJ/ol) enough residual internal energy (~1 ev) to break into fragents 70 ev is so large. Reaining is used to break olecules M (/z 6) very rare, /z 197,

3 -1. What is MS?.5 For HCHO, lower the energy less fragentation Base peak: ost intense peak -1. What is MS?.6. Cheical Ionization (CI) : less fragentation than EI : use reagent gas for ionization CH 4, isobutane, aonia, ~ 1 bar CH 5 e- (100-00eV) convert CH 4 CH4 4 CH e CH e 4 CH4 CH5 CH3 CH4 CH3 H 3 CH4 CH5 H CH Proton donor ro analyte olecule M C H 5 MH CH4

4 -1. What is MS?.7 3. Resolving Power R.P.= Δ or 1/ Higher, the better to separate two peaks with siilar ass - Oh, Mass Spectru, Speak to Me.8 Each ass spectru has a story to tell. Nitrogen rule : a copound has an odd # of N M odd noinal ass even # even 1. Molecular ion & Isotope pattern EI for aroatic copound usually base peak M M1 provides inforation on eleental coposition : follow the natural abundance of isotopes Table -1

5 .9 - Oh, Mass Spectru, Speak to Me.10 Intensity of M1 : n x 1.08% x 0.01% for C n H fro 13 C fro H For benzene: C 6 H 6 M : /z 78 /z 79 : 6x1.08 6x0.01=6.55% Check with Fig -6 Cl, Br: nearly equal peaks at /z 136, 138

6 - Oh, Mass Spectru, Speak to Me.11. High-Resolution ass spectroetry C 5 H 8 O = Da C 6 H 1 = Da With double focusing MS Or accurate TOF MS Resolve at /z 100 All with noinal Mass=31 /Δ= Oh, Mass Spectru, Speak to Me.1 3. Identifying the olecular ion peak How do we know /z 100 olecular ion? M 1) : highest /z value but 5~0% intensity of the base peak ) M1, M ratio 3) Heaviest fragent should not correspond to iprobable ass loss fro M loss of 3-14 or 1-5Da? No way CH 3 : 15, OH, NH 3 : 17 H O: 18, C H 5 : 9 OCH 3 :31, CH 3 CO:43 /z 100, next highest /z 85 (-15: CH 3 )

7 - Oh, Mass Spectru, Speak to Me Interpreting fragentation patterns How any fragent ions fro -hexanone? -3 Types of Mass Spectroeters Magnetic Sector MS -- Figure - Resolving power = ~0~1000, 0.1 at /z 100 ~ 10 5, at /z 100 (double focusing) Double focusing: ion passes agnetic sector & electrostatic sector

8 -3 Types of Mass Spectroeters.15. Transission Quadrupole Mass Spectroeter GC-Q: ost coon cheap analyzer, vac ~10-9 bar by turbo olecular pup, Ion acceleration : 5-15V E field deflects ion when igration. Allows only one /z ion to reach detector by applying constant V & RF oscillating voltage Up to 4000/z in 0.3 /z constant Resolving power : , Types of Mass Spectroeters Tie of Flight MS (TOF-MS) ,000 ties/s, 5kV to accelerate ions kinetic energy of ions 1 KE = v Sae KE but diff.ass Different flight tie REFLECTRON SYSTEM : iproved resolution

9 -3 Types of Mass Spectroeters.17 Reflectron syste : ions of different ass ay have slight difference in KE ay have sae flight tie. No good in resolution at hollow rings with increasing positive potential, - the last with ore positive than accelerating potential : Repelling Ions slow down, stopped and reflected back to left. all ions of sae /z sae flight tie : R s = 1000~5,000 (accuracy ~0.001) : but low pressure 10-1 bar : high acquisition rate - 10 ~10 4 spectra/s to /z Types of Mass Spectroeters Quadrupole Ion-Trap MS (TOF-MS) : ost widely used MS, : interfaced with chroatography colun constant DC-V, RF voltage to central ring electrode causes ion circulation increase aplitude of RF-voltage expels ions of a particular /x (unstable trajectory) passing through exit holes detection scanning /z 10~650 can be 8 ties/s Resolving power = 1000~4000, /z 0.1 acc. ax /z ~ 0, ties higher sensitivity than T-Quadrupole

10 -4 Chroatography-Mass Spectroetry.19 GC: capillary GC colun directly to MS EI or CI used LC : volatile obile phase additives needed spraying device by pneuatic or atospheric pressure cheical ionization 1. Electrospray ionization (ESI) : for liquid saple ESI: pneuatically assisted electrospray -4 Chroatography-Mass Spectroetry.0 Electrospray ionization (ESI) Nebulizer: 0V Spray chaber: -3.5kV : positive ion ode fine aerosol vaporize ionization MH (M1) M(NH 4 ) (M18) M(Na ) (M3)

11 -4 Chroatography-Mass Spectroetry.1 fine droplets : ~1μ by solvent evaporization repulsive force = cohesive force of surface tension. in ESI, LITTLE fragentation of analyte siple spectra for fragentation? collision induced dissociation (CID) between capillary and skier cone -4 Chroatography-Mass Spectroetry.. Atospheric Pressure Cheical Ionization : APCI : heat & coaxial flow N convert eluate into a fine ist like CI, gas phase reaction creates new ions fro gas phase reaction between ions and olecules : under High voltage to a etal needle, Corona (plasa containing charged particles) around needle. inject e- into aerosols ionization protonation

12 -4 Chroatography-Mass Spectroetry.3 N N N 4 H O H O 3 H O 3 e N N H O H O H O H O nh O H O (H O) N n e 4 N 3 3 N M MH OH (H O) n Or (n 1)H O M e X Y e Y M X AH A Y HY Adv: : Handle variety of analyte : Upto ~ L/in : but only singly charged ion Disadv: : Not good for acroolecules. : Little fragentation -4 Chroatography-Mass Spectroetry.4 3. Selected Ion Monitoring (SIM) & Selected Reaction Monitoring (SRM) To increase selectivity & sensitivity of MS by decreasing the response to everything else Chroatogra (UV) of Herbicide spiked in river water TIC

13 -4 Chroatography-Mass Spectroetry.5 In SIM, only a specific /z is onitored. S/N is greater -4 Chroatography-Mass Spectroetry.6 In SRM, triple quadrupole MS Q1: select one ion Q: collision cell, N or Ar : ~10-8 ~ 10-6 bar Q3: select only specific product ions to detector

14 -4 Chroatography-Mass Spectroetry.7 ESI-tande MS of pure estrogen EE By triple quadrupole MS EE : (17a-ethinylestradiol) : contraceptive Ppt level reproductive disturbance in fish SRM chroatrogra showing the elution of 3.6 ngl of Huan estrogens (horones in ovarian cycles) extracted fro in sewage water. Signal : the su of /z fro Q3. -4 Chroatography-Mass Spectroetry.8 4. ESI of proteins : ESI well suited for charged acroolecules ass M n(1.008) M To find charge, /z= n n = = = charge n n The next peak at lower /z should have (n1) protons M (n 1)(1.008) M = = n 1 n 1 n 1 n M/ n n 1 = = M/(n 1) n Charge: n = 1 n ass of neutral olecule M = n ( ) ESI-MS of cytochroe C Mass can be easured. Instruent calibration with known ass proteins

15 -4 Chroatography-Mass Spectroetry.9

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