Mass Spectrometry and Chromatography
Mass Spectrometry and Chromatography CHEM 5181
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Mechanisms of Ion Fragmentation AcLa erly Chapter 4 CU Boulder CHEM 5181 Mass Spectrometry amp Chromatography Prof JoseLuis Jimenez Fall 2007 Interpretation Lectures vs McLafferty Chapters Ch Ch Ch Ch Ch Ch Ch Ch Ch oh 1 17 7 8 9 l 9599 h troducti on Elemental Composition The Molecular Ion This Basic Fragmentation Mechanisms Zoday course Postulation of Molecular Structures 1 Atmiliaif f Tec lm iques Theory of Unimolecular Ion Decomp Detailed Fragmentation Mechanisms AdVa ced I Vers1on Fragmentation of Compound Classes 0 Computer Methoeiis Introduction to Fragmentation Reactions Earlier the mass spectrum shows the mass of the molecule amp the masses of pieces from 1t 0 Additional information ion abundance 7 Relative abundance of an ion can be an indication the structure of the fragment and its environment in the molecule 0 Source Unimolecular iondecomposition reactions 7 Another branch of chemistry 7 Not completely understood or predictable 0 Study spectra of closely related molecules 7 MS not sensitive to all structural features 7 Many close similarities to pyrolitic photolytic radiolytic reactions as well to condensedphase organic reactions 7 But here each reaction involves ions amp often radicals under vacuum 7 Rearrangement reactions are possible Catchup on Organic Chemistry You ll probably find this part easy if you 1377MB have a strong org chem ANEUAGE background Organic 39 If you don t you may I need some catchup reading before you Transla ng the understand McLafferty Basic Canning Eg book by Klein Org Chem as a second language Unimolecular Decomposition Reactions 1 EI MS reactions are unimolecular as opposed to other techniques such as laser ablation ionization M4quot are made with a Wide range of internal energies 7 Cool M4quot Will not decompose 7 ABCD e39 gt ABCD t Unimolecular Decomposition Reactions 11 Excited or Hot M4quot Will decompose in a chain of energydependent reactions 7 Now things get interesting 7 Each one with a neutral loss 7 Rearrangement NO r from NH4ZSO4 e ABCD gt ABCDquot Au BCD gt A 300 Bc D 7 D ABC A Bc 7 AD BC Site of Initial Ionization I I From Smith amp Busch sect 32 I IIIIIIE 0 e39 most vulnerable to ionization are those of highest energy HOMO highest occupied molecular orbital Also form the weakest bonds IP is de ned as the energy to electron n gt TE gt o n lone pairs from 0 0H 0 00 remove the weakest bound E0 heteroatmoms Hcu4 Rzng 233H2 0 0 0 0 OH tilt Figure 32 Relative energies of molecular orbitals in organic compounds Site of Initial Ionization II ngtngto OI CH3 quotquot CH2 CH3 I IIIIIIE 45 CH3CH2 CH3 Figure 34 Examples showing notation for localization of initial ionization site I From Smith amp Busch sect 32 l Factors that In uence Ion Abundance I 0 Most important Stabilitv of the product ion Electron sharing stabilization From nonbonding orbital of heteroatom n CH3CO lt gt CH3CEO mz 43 Resonance stabilization CH2CHCH2 lt gt CHZCHCH2 mz 41 Benzyl C6H5 gtgt phenyl C6H5CH2 Distonic radical ions Separation of charge and radical sites CH3CH2CH2CHO gt CH2CH2CH2CHO H CH3NH2 gt CH2NH3 Factors that In uence Ion Abundance II Stevenson s Rule In a cleavage of a single bond in an OE 39 ABCD can give A BCD or A BCD The fragment with the higher tendency to retain the unpaired electron should have the higher ionization energy converse true It will be the less abundant ion in the spectrum Factors that In uence Ion Abundance III IIIEIEII Loss of the largest alkyl CnH2n 1 Exception to Stevenson s rule abundance decreases with increasing ion stability CH3 cHa CH3 csz CH cwg39 gt cszcH gt CHC4H9 gt I Ha CZHSCHC4H9 gt cszcc4HQ Factors that In uence Ion Abundance IV IIIIIn Stability of the neutral product Stability of ion is much more important A favorable product site for the unpaired electron can provide additional in uence 0 Electronegative sites such as oxygen OR The neutral product can be a molecule 0 Small stable molecules of high ionization energy are favored H2 CH4 H20 C2H4 co NO CH3OH st HCl CH2CO and C02 Losses of 2 16 18 28 30 32 34 36 42 44 Factors that In uence Ion Abundance V EEEEEEEEUUH Entroy Steric Effects Most favored reactions for enthalpy often have steric restrictions e g rearrangement Dissociation favors products with less restrictive entropy requirements even if the enthalpy barrier is higher ie a simple bond cleaveage 0 Unknown 4 l Predict the most abundant ion in the spectrum of Factors that In uence Ion Abundance VI EEEEEEEEUUH Cleaving CHZCH2 bond is sterically easiest Paminobenzyl cation is very stable via resonance Reaction Initiation at Radical or Charge Sites IEEEDDD Fragmentation reactions are often initiated at the favored sites for the unpaired electron or the charge The most favored radical and charge sites in the molecular ion are assumed to arise from loss of the molecule s electron of lowest ionization energy Favorability 039lt 7zlt nelectrons see example spectra Sigma 0 RHzCICHR39 33 RHQC39CH2R39 Pi1t RHCIICHR39 3 RchtCHR39 Nonbonding n R O R39 i R O R39 Unlike M charge localization is implied 15 Reaction Classi cations I IEEDDD Decompositions of odd electron ions 1nvolv1ng s1ngle bond cleavage results 1n an even electron 1on and a neutral rad1cal Stevenson s rule applies Reminder in CXHyNZOn RPDB xl2yl22l yz is odd gt RPDB ends in l2 gt EE yz is even gt RPDB Whole gt OE 16 Reaction Classi cations II IZIZIIZIIZIIZIEIEIIJ Decompositions of odd electron ions involving two bond cleavages can results in an odd electron ion and a neutral Rearrangements Decomposition of rings HZCCHOH HZCZCHOH HZCZCH2 charge retention H c 5 2 2 HQCZCHOH H2CCH2 charge migration 44 0 OH HOH HZCZCHZ charge retention H r P Hzc H2 HOH H2CCH2 charge migration Reaction Classifications III IIIIIIIIIIIIIEIIII Cleavage of three bonds in M or any OE produces an EE l 74 CHscHz CHscH CH30H2 02H3 H2 N H Reaction Classi cations IV IIIIIIIIIIIEIEI Evenelectron rule decompositions of even electron ions typically result in another even electron ion and a neutral Odd electron formation is not energetically favorable CH30HZ OCH2 charge migration CHCH2 CH2 gt CH2CH2 HOCH2 rearrangement charge retention CH30H239 ocH2h quotCH H20 CH CHz CH CH2 2 charge retention unfavored 46 HzC CH2 CHQZ H CH2CH unfavored l9 Reaction Classi cations V IIIIIIIIEIEI Table 41 Types of ion decompositions Product iona Precursor Number of Charge Charge ion bonds cleaved retention migration OE39 M39 1 EE or EE i OE39 M39 2 OE39 cm OE39 oci OE39 M39 3 EE orator EE acct1b EE 1 OE L39b EEJr EE 2 EE r OE L39b aDesignations a and are alpha and inductive cleavages respec tively as explained in the text Two ireactions lead to the same charge behavior retention or migration as two or reactions Brackets indicate products of reactions discussed in Chapter 8 b Not favored OE formation from fragmentation is only favored for cleavage of two bonds of precursor OE This is Why we mark important OE ions in spectrum 20 SigmaBond Dissociation 7 IIIIIIII I Eg alkanes Every valence electron is shared in a bond A bond nds itself with lelectron breaks Alkanes R CII3 43 R CR3 Ionization of CZH6 increases CC bond length by 30 halves its dissociation energy Fragmentation is favored at more substituted C u e CH3CCH20H3 M CH33CCH2CH3 1 CH33C CH20H3 100 11 Relative intensity Relative intensity IIIIIIII Alkanes amp Branched Alkanes 100 a 22 Only 1mportant OE is M so as a C3H7 amp C4H9 are a 85 most stable L L5 i1 Ll I i 1413 127 141 115 17 F 088 Of H2 and H mz 2 0I I I4390I I I610I 8390l I 1301 1101 I l1ampll0I I 1amp0 I 1amp0 Figure 32 Mass spectrum of dodecane lt lt 1001 127 716 w Fragmentation 51 50 at subst1tuted C g as new Loss of the larger L5 ll I ll AL 39 113 1iquot 111 155 i alkyl I I I I I I I I I I I I I I I I I I I I I I I I I ITFTI I I I mz 20 40 60 80 100 120 140 160 180 Figure 33 Mass spectrum of 4methy1undecane 22 RadicalSite Initiation oncleavage I IHIIIIIIEIEIIJ Unpaired electron at radical site has strong tendency to be paired Donate unpaired electron to form new bond Need a 2nd electron take it from bond of adiacent C atom 0c carbon a o CHs T39CHZ OCZHS quot gt 39 CH3 CH22002H5 6 CH2 0C2H5 50 c0H3 100 Fishook arrow is movement of single electron Same as homolytic cleavage of organic chem Only radical site moves stays 23 RadicalSite Initiation oncleavage II IIIIIIIIIEIEIIJ Tendency of radical site to initiate reaction Parallels tendency of radical site to donate e39 NgtSO7tRgtClBrgtH But it is affected by its environment in molecule 0 Unknown 42 what will be the most abundant fragment of HOCHzCHzNHZ 24 RadicalSite Initiation Qtcleavage III IIIEIEIEI Carbonyls ICE395 r 0 2920 WEI w 02115 CZHSmCE 02115 100 Doublebonds I 00 on CHSWCHZwCSHS gt CH3 39 CHZZCSHS quot399 6H2 CGH5 100 415 Loss of Largest Alkyl in occleavage IIIIIEIEI CH3 X a 03H7 x1011 CZH5CCH3OH gt C3H7CCH3OH gt I I 3sz mz 73 100 mz 87 50 103H70102H5gt6H1 mz 101 10 1 3943 9 CH3 C3H7 C OH 3 15 I 395 C2H5 C 9 73 E 50 810 g 27 E 55 a 0 116M 1Q10L 115 J In lllll l l i I I I I I I I I I 39 I I I I I I I I 39 l mz 20 40 60 80 100 120 Figure 42 Mass spectrum of 3methy1 3hexanol 26 Relative intensity ccCleavage of Aliphatic Amines 39 Very dominant due to e39 donating abil 100 55 4 THB quotquot CH3quotII TNH2 50 IE CH3 1L 0 41 73M j I Il III I I I I I I I I I I I I I mz 20 4o 60 80 CHg I I AA I I I I I D I It I b H W IEIIJ E2 E3 EJD ity of N 07 5 NHCH20H3 H L52 1007 53 3 39 5 C 52 E a 50 2 E 73W a ct 15 III LI IIIIIIIlll mz 20 4o 60 80 Spectra of Isomeric C4H11N Unknown 44 Unknown 45 100 30 100 30 a g E g E E g 5 g 50 E I E 73 73 15 Illll 1414 15 I l l lllIIll IIIIIIIIIITT mz 20 4o 60 80 mz 20 4o 60 80 39 Structure of 100 58 each t 9 5 spec rum g 50 g 2 42 73 30 1 JhI all I I I I I I I I I I I mz 20 4o 80 Unknown 46 IEIIJ E2 E3 EJD 100 58 g w C 8 E a 50 2 U 75 3O 0 a 43 73 115 III III II I IIIIIIIIIIIIj an 2O 4O 60 80 Unknown 48 100 44 g w C B E a 50 2 E Q D 29 58 15 J I III I hI 7 IIIIIIIIIIIII mz 20 60 80 ChargeSite Initiation icleavage IIIIIIIIIIIEIIJ Inductive cleavage heterclytic dissociaticns 0f OChem For OE 1 j R YFl W R YR R R W 0 q o l 0 gtY H 3 Y LA El R CZY R R Example We i C2H5wOCZH5 quotquotquotquotquot CZH5 OC2H5 40 i cleavage for Aliphatic Ketcnes R R 3pentan0ne a FZB 6 6 I R RCO and 3 methyl 39 2butan0ne R R Which is Which Unknown 49 Unknown 410 100 29 57 100 43 Z a i E 50 50 E 86M39 g 15 27 86M 1 e 1 15 l 7 L IIIIIIIIIIIIIIIII IIIIIIII1IT7 8 a a E a 3 a a Decompositions of Cyclic Structures I llllllll mz 84 mz 84 mz 56 39 430 Breaking one bond we don t 50 get fragments 0 Need to break at least two bonds I I ii I I i I I39i I I Create an OE mz 40 31 Relative intensity Decompositions of Cyclic Structures 11 IIIIIIII R R Ix A I Create egt gt R distonic gt a charereen ion rad1cal 1011 a I lt g H I 6 mz 54 R H 80 05H7 100 39 RGU O DIGlS ill IE 90 RCeH5 04 431 99 Alder R0 Rw j gt charge migration A N R H IE 105 lt 5 100 N R 05H5 IE 84 100 104 Relative intensity 158M 78 91 115 15 27 39 69 II I il 1 19 113 I I I I I I I I I I I I I I I I I I I I I I mz 20 40 60 80 100 120 IIIIII 140 160 32 Radical Site Rearrangements I IIIIIIIZIEIEIEI 039 McLafferty Rearrangement Unpaired e39 donated to form new bond to adjacent atom 211d electron comes from adiacent bond that breaks charge retention p H i H R U 0 rH R 2 0 0 R HO M TI age rd v o m258 R CH3 40 IE 87eV R CSH5 500 433 H H M Q k l R CH3 IE 97 5 R 06H5 IE 84 100 charge migration 434 i Radical Site Rearrangements II IIIIIIIIIEIEI 43 100 VYO 50 86 27 41 58 71 15 29 39 I l 87 C 39393939lli39 li3939i39393939i39393939i3939 l I I 10 20 30 4O 50 60 7O 8O 90 100 m ainlib 2 Pentanone mZ 58 formed by McLafferty rearrangement 0 As charge retention in previous slide RH 34 Lecture Cl Part 2 Introduction and Theory of Chromatography CU Boulder CHEM 5181 Mass Spectrometry amp Chromatography Prof Jose L Jimenez Fall 2007 Reading Braithwaite amp Smith Chapters 1 amp 2 Review Clicker Question In chromatographic separations A Equilibrium is always reached at every position along the column B The solubility of the analyte in the stationary phase is the most important parameter C The kinetics of mass transfer play an important role D A and C I don t know m Concept of Peak Capacity a Time or Area Available for Separation Time or Area of an Individual Peak Fig 4 Memmyemass plul Ufa cumplEX mixture eun39ammg multiple 5 e ads ebsem a Linesare Supenmpused emu theplut m meme the mnbunye mass trends re each class er uleeule wquSrMSfurpepudesZ arerLcancRm x m7 s 5P 1 GI Ingzzlz 6 Stem mdDzwdH Elise Immol39clmmmgwhy m 172232m2 Pigs E5392 m de ux atle mm mumnm mssso Sgarating EfficiencLi Peak Width 11 Described by variance 0392 units s2 adetermined from Gaussian fit to peak last ecture 7 l Classical chromatography theory 7 Separation in Ndiscrete steps plates Height Equivalent ofla fPlate lllllllllllll Height Equivalent to One Theoretical Plate HE T P H i N Application Calculate H and N for peak 18 assuming a 60 m column A H 5 pm B H 50 um C H 500 pm D H 5 mm EId0n t know 33 Separating Ef ciency Peak Asymmetry IIIIIIIIIIEIEI Normal peaks 39 Q What can cause peak asymmetry h h False retention times a b Figure 24 Peak asymmetry a Fronting and b tailing 34 Normal peaks 39 2 h h False retention times a b Figure 24 Peak asymmetry a Fronting and b tailing I Separating Efficiency Peak Asymmetry T ailing some part of the stationary phase binds analyte molecules more strongly F ranting some molecules move ahead inject too much sample gt saturate Stat Phase 0 Peak Asymmetry 19 AS at 10 h a 09 lt AS lt 12 for acceptable chromatography Equations for Calculation of Chromatographic Figures of Merit for Ideal and Skewed Peaks J P Foley and J G Dorsey Anal Chem 55 730737 1983 35 When there is a gradient in concentration of a species that can diffuse in medium dCA D JA A dy j A molecular ux of A moles cm392 s l o C A concentration of A moles cm 3 o D A B diffusivity of A in B cm2 3391 01001 cm2 3391 in gases 10395 cm2 5391 in liquids Diffusion Fick s lSt Law I I I III III III El III Concentration y r as u ubmsod A A 39Concentration y 7 as UIUi1SOdA A 36 From Bird Stewart amp Lightfoot Transport Phenomena 2nd Ed 2002 Mass Transfer Kinetics Fick s Law IHIIIIIEIEI 0140 Initially no substance A Thickness of Pulse at the surface slab of Stat Phase y tlt 0 substance B l Transient concentration Steady state pro le at long times A on WAy mass ux of A wA mass fraction of A wA y t Smallt DAB diffusivity ofA in B L S surface area 0 density P de A y dy j Av molecular mass ux of A Large t jAy IODAB RV 0140 JOion I Situation in Column Qhromatography Il I E DA 2 O 01 8 um Thickness of i slab of Stat Phase Y t lt 0 t 3 substance B l t 0 N t Z 4 DA on KW g kl I wA y t i r 1 t Z 5 i I i 601 1 I 5 5c M m DAG 39 Am Mass transfer takes time gt separation limit on resolution Clicker Question 0 When an analyte is diffusing in the stationary phase equilibrium will be reached faster A When D A is small B When DA is large C When SP thickness is large D E A andB A and C 0 Conservation of mass for diffusing species in control volume Diffusion Fick s 2nd Law y y Per umt area y I perpendicular to diffusion 6C t 2 Jm M at aCAy1t Ay D aCAy1t D aCAy1Ayt at 8y 6y Concentration y Diffusion Fick s 2nd Law II Concentration y 0 When things are changing in time 3 S t A ay2 3 0 Once CA is the same everywhere we have reached equilibrium in the SP Q can we estimate orderofmagnitude of time needed 41 Time Scale of Mass Transfer Concentration y For transfer across SP thickness Y 39 Start with D at A 2 39 Orderoflmagnitude analysis as u Housed K ACA D ACA A 2 TD Y Simplifying T Z Y2 D DA 42 Numerical Example of Mass Transfer IIIIIII 0 Assume Column diameter 100 um Film thickness Y 1 um Diffusivity of analyte in stationary phase DAB 10396 cm2 s1 Solubility of A in B is 1 of volume MWA 100 g mol391 Questions What is the time scale of mass transfer What should be the time scale of ow along 1 mm of the column What is the max amount of analyte that can be in the stationary phase per 1 mm of column length Film thickn 01 8 um SSS 43 Processes HZIZHZIZIEIIZIEIEID Band Broadenig Mobile Phase 0 Noncolumn broadening Dispersion of analyte in Dead volume of injector Connection between injector amp column Connection between column amp detector Emphasis on minimizing dead volume injectors ttings 0 Column broadening Van Demteer model Schematic of Column Chromatography III IIIIIIIIIIIEIEIIJ AA AA A A A A A A AA AA AA AAA AAA AAA 0 If analyte has some affinity to the stationary phase it Will be retarded mmmmmm KZQ Kinetics CM 0 Molecular mass transfer diffusion Emerge at the detector after retention time tR 45 IIIIIIIIIIIIEIEIIJ Kivsl jCohmnm Substrate Partlcles r W o 039 Film thickness W O 018 um Most GC columns do NOT have particles Most HPLC columns do have particles Why Particles are needed to prevent liquid ow for being too fast Particles are needed in HPLC to shorten diffusion distance in MP Particles are not needed in GC because diffusion is very fast B amp C I don t know muowgt 46 Effect of Mobile Phase velocity on H IIIIIIIIEIEIIJ 04 L1 uld chrom t ra h Experlment a q g p y 03 Repeat the same 5 02 separat10n same a column and mobile 0 1 phase I I 0 05 10 15 H Linear ow rate cms b Gasliquid chromatography Observe an optimum H g 60 r 50 1ncreases to both s1des m 40 30 Goethe there IS I I I j 0 20 40 60 80 noth1ng more pract1cal Linear ow rate Cm 99 than a gOOd thCOry FIGURE 24 7 Effect of mobile phase flow rate on plate height for a liquid chromatography and b gas hr C omatOgraphy I Skoog amp Leary 4th Ed I 47 Van Deemter Model fA Term Broadening Eddy diffusion amp unequal pathways 0 Molecules may travel unequal distances 0 Particles if present cause eddies amp turbulence 0 A depends on size of stationary particles want small and their packing want uniform or coating in TLC plate GC 150 um HPLC 510 Hm A MP Clicker A Aterm iasu i 1 B Atermiasu i he Ho 9 C Aterm 7 f 17 1 1 i OEQDOBQQ v9 D Don t know Fig 311 Illustration of eddy diffusion 48 Van Deemter Model B Term IIIIIIIIIIIIIEIEIIJ t t 0 Longitudinal Diffusmn Sggfggfrg g Forward and backward diffusion in mobile phase Bas1cally molecular d1ff as if as band moves along wquot A mobile phase was not moving mtg o 7 ClleCI 1 Analyte band A BtermTasuT B BtermiasuT C B term 2 f0 D Don t know Concentration gt Clicker 2 B term is A more imp in GC B More imp in HPLC 0 Similar importance Fig 38 Bonn onooooninn on in noioonion oiiinnion iinoo iinon ninoono n gt o gt D I d 9t k From Miller J M Chromatography Concepts and Contrasts John Wiley amp Sons Inc New 39 York 1987 p 31 Reproduced courtesy of John Wiley amp Sons Inc 49 Distance along z axis Van Deemter Model B Term IIIIIZIEIEIIJ Model for B B ZyDM 0 y is hindrance factor due to packing 07 ir packed l in open and DM is molecular diffusion coeff B terms dominates at low u Enoyoiiiiioion Molecular diffusion 50 Van Deemter Model C Terms 1 IIIIIIIIEIEIIJ Movement onto SP Mobile Accounts for finite time phase A C Stationary phase SP Analyte attracted onto SP Movement off SP for mass transfer equil btw 7 analyte in stationary and mobile phase not instantaneous Most important effect in GC amp HPLC C 5 accounts for stationary phase mass transfer df stationary phase lm thickness d2 DS diffusion coeff of analyte in SP CS f Thinner lms reduce mass transfer time DS amp broadening But also reduce capacity of the column 51 Van Deemter Model C Terms 11 IIIIIIIIIIIIIIIEIEIIJ C M accounts for mass transfer on the mobile phase interface with the SP 0 In packed columns d2 CM P dp 1s particle diameter DM 0 In open columns C 2 d5 M dc 1s column diameter DM Clicker 1 Clicker 2 CM term is A C term T as u T A More important in GC B C term 1 as u T B More important in HPLC C C term 7 f7 C Similar importance D Don t know D I don t know I Van Deemter Model of Band Broadening lIIIIIIIIIIIIII 0 Tries to explain previous experiment L 0 H plate height 17 tM 0 5 average linear velocity B H ACSL1CMLI u H as small as possible calculate Hmin 0 Some terms decrease other increase with E There should be optimum 0 There are alternative models see reading Optimum Mobile Phase Velocit IEE DUDE 0 We want N highest H lowest Optimum Mobile Phase Velocity GC amp HPLC IEIZIEIZIIZIEIEID H mm b H mm N2 WCOT 03906 39 0 ltlt 004 1 9 002 4 25 50 75 05 390 115 U cm 54 U 0m 8 1 Figure 27 Van Deemter plots of plate height H against average linear velocity 0 of the mobile phase a the contribution of each term to the composite curve b plots for WCOT GC columns using N2 He H2 carrier gas and N2 for packed column GC PGC 0 plots for HPLC and SFC composite curve Q differences in A B C between Supercritical Fluid Chrom amp HPLC 55 Lecture 2 Ionization Techniques Part I CU Boulder CHEM 5181 Mass Spectrometry amp Chromatography Prof JoseLuis Jimenez Fall 2006 J 10 Mass Detector Source quot1Analyzerl 1 Fourier Transform in Igor Ion Sources Neutrals don t feel electric or magnetic fields Once molecules are ionized they immediately feel the forces Ion sources use electric fields to steer ions into mass spectrometer Example of an E1 Ion Source Clicker Q A ion Will go faster thm the base plate than thm the quad Will go slower thm the base plate than tth the quad Will have the same velocity in both cases gt 4m Wm 031 O D It doesn t have enough energy to enter the quad P1 I don t know pmmmumunmmummmmmmsmmm 4 An Einzel Lens mm mm web in gure A remrdmu Emzel lens hm hm mee tubular elenrones held sx duffvrcm paten bls e mam 0Hch at m mzel lens Shawn 45 mm um um velaclly s dvashnzllv decreased in me llrsl In of Ihe lens and hen drashcally lncveased a air in me second haw rhe deceleva ng and ck news which coneehum mm upli raglan olmelr Note he be close in me pelenllal a lm rivathelons own cal axlsan usale acusmg e verall ellecl ol these owes is Indicated by lhe hollow avruws lnce mess mes toward and away mm the apxical aXlS aw payable magnitude they are must eeme in ms middle when we ahs she slaw and consequently spen mast nme Fm ms reason we Iehs shown is overall lowslng r2 also hm xhe manual of he middle elgcvmde mun me m some a me lens s m be Ion Optics Simulation Fully deterministic can be realistically simulated But quality of your ion beam Width angle energy matters a lot Einzel Lens httpwwwviasorysimula onssimuso iionop cs html Lfyou ever get serious use SIMION httpwwwsimioncom quot Re ectron Ionization Techniques that we will discuss EEEEEDDD Electron Ionization EI Chemical Ionization CI Electrospray ESI Nanospray Desorption Techniques Fast Atom Bombardment FAB MatrixAssisted Laser Desorption Ionization MALDI DESI DART Ionization for Elemental Analysis Thermal Ionization Source Spark Source Glow Discharge InductivelyCoupled Plasma ICP Q why are so many ionization techniques used in MS Ionization Methods Characteristics IIIIIIIIIEIEIIJ TABLE 135 Desiderata for Ionization Methods Characteristic Desired Characteristics Usually Achieved High signals ion current High efficiency neutrals gt ions Bipolarity positive and negative ions High molecular weight compounds All sample states Control of fragmentation 103910 A approximately 109 ionss 01 Except for El Approximately 105 Da All physical states are compatible with MS By control of energy deposited in ion Not every method exhibits all these characteristics even for the most favorable analytes From Lambert Effect of Ionization Techniques me Schewdt 5 MN m 166 an 3 60 Ac 1 0 sz 2quot a H NCH 0 heldlammliontFl O H 3 helddasuprllnnllD A 0 m7 ephedrine MW165 ma Mu m i 65 BK E 50 ea 5 40 40 i 5 20 92 45 20 E cr n l U n on 50 mz n so mo lt50 mz K wummi m u 39 39 39 q Same molecule analyzed by 4 techniques Information is complementary use gt1 technique if possible Goal for today understand Why this happens Electron Ionization Source Scheme Fxtracti on Electron Collector Electron Ionization Source EEEEDDE FROM FF INLET O SYSTEM ION SOURCE HOUSING SAMPLE MOLECULES IN VAPOR STATE ION FOCUS PLATES TO MASS ANALYZER o IONIZATION CHAMBER o o o ACCELERATION PLATE VACUUM From Watson FIG 71 Schematic diagram of electronionization source 11 El Notes 1 1EEEEDDE 0 Hot lament giving off electrons Thermoionic effect W or Re filament Accelerated by a potential difference towards and anode Interact With the gaseous molecules in their path Do not impact them ions formed Ionization Efficiency E molecules present What characteristic of the electron can we change to try to improve the results of ionization Electron Interaction Cross Sections SF6 Electron Energy eV 103 I I I It I I II 07 I TotaI Scattermg E asuc megra1 o 102 Attachment 0 Momentum o S 101 C 9 6 10 D lt0 3 10quot 1 39 9 MIMI O 39 I I 10 2 I I DIssocIatIveAttachment 39 103 I I I l I I I 0001 001 01 1 10 100 1000 Electron Interaction Cross Sections CF4 mp UphysIcs mst guvDIvIsIunsDIVBAZHeamdaIzFDFlDatabaseschnstu pdf I I I In lmcl VIblabanal NE 10 quot o h o 5 10 x g 14 g IV EIasmImegIaI I I I 13 1039 mwvma ma ToIchmzaIIun h CF 0 10392 1 TcIaIAIIachmemA aw I I I I I I 001 01 1 10 100 1000 EecIron Energy eV Figure 1 Ell39Cll39llilll lquottll l ion cross wrl ions for CFI I Ionization Ef ciency ys Electron Energy 1 IIIIIEIEIIII It IJ From C Watson Ionization Potential log of Ion Current 1 IO 20 30 electron energy eV 70 FIG 73 Relationship between ion production and energy electron volts of ionizing electrons region A threshold region principally molecular ions produced region B production of fragment ions becomes important region C routine operation mostly fragment ions 15 Ionization Ef ciency vs Electron Energy 2 IIIIIIIIEIEIIJ From Hoffmann L O L 9 Number of ions produced per cm free path length and per mmHg sample pressure 72 10 10 50102 108 104 Electron energy eV Figure 12 Number of ions produced as a function of the electron energy A wide maximum appears at around 70 eV 16 The Concept of Cross Section Electrons are coming perpendicular to page OCGDQ Physical Scattering 70 eV 15 eV Cross Cross Ionlzatlon Ionization LOW Section Sect10n Cross Cross Section Energy SeCtIOH Attachment eg SF6 Time Scales of Ionization 0 What happens to the molecule when an electron goes by 70 eV electron gt 5 X 106 ms Molecule 10 A 1 nm Transit time 2 X 103916 s Molecular Vibrations gt 103912 s Electronic time scale N 103916 s 9 FrankCondon principle nuclei remain frozen in position L gt10 12 5 gm 10 5 s L0gt 18 ecayquot EI Notes 3 What electron energy would be most interactive with the molecule Each electron is associated with a wave 7 h mv 27 A for 20 eV 14 A for 70 eV Wave is dispersed into many frequencies If one of them has an energy hv corresponding to an electronic transition in molecule energy transfer leads to excited electronic state 7 10 to 20 eV are transferred to the molecule Only 10 eV are needed to ionize so rest of the energy can lead to fragmentation Ionization potential energy it would take to eject the weakest bound electron from the molecule At very high energies the wavelength becomes too small and the molecules become transparent to the electron In other words not enough time to interact transfer energv 19 Energy Balance of El eV 70 Q is energy conserved Internal Energy Distribution after E1 lllllllElElll From Lambert F l Molecular ions Those of low energy are stable to spontaneous dissociation ExcHed Neutrals Iquot nf l I 70IEeV i IE gt 6 eV Figure14 28 Stylized internal energy distribution Pe resulting from Impact by electrons of 70 eV kinetic energy on an organic molecule The maXImum internal energy that an ion can acquire is 70 eV minus the ionization energy llE Most ions acquire small internal energies zl Illllzlll Soft and Hard Events 1 Seitlemzatmnjlm e e EMT am C 39 e gt Stable H H E e39 e 3 gt Fragments e SE 2 Hard Soft Events gt Fragments Figure 133 Electron ionization accompanied by different degrees of excitation of the molecular ion Soft ionizing events transfer little excess energy to the ionized molecule which is observed intact Harder collisions also occur and give rise to the fragment ions fre quently seen in El mass spectra See also http schwingercaltechedu Needionizationhtrm Soft and Hard Events 2 Illlllllll From Lambert hard collision onset of fragments M 5 ft collision Potential Energy gt M Molecular Coordinate gt Figure 135 Vertical transitions associated with electron ionization M gt M39 showing deposition of internal energy a in the ion Mt Two ionization events are shown a soft collision leading to stable Mt39 of relatively low energy 81 and a hard collision leading to high energy molecular ions 82 which rapidly fragment Fragmentation notes Illllllllllll Fragmentation depends on Internal energy deposition on the ion Shapes of the potential energy hypersurfaces Energy of the interacting electrons Molecular structure resists fragmentation Chemical nature of the analyte 0 Is fragmentation good or bad E1 Fragmentation vs Electron Energy Q why is there such a dramatic difference in the spectra I I E E E E E E I O H From Choker Q Hoffmann 1 r t 42 v0 39 70 eV S Opera 1011 a g g 2000 O NCH3 low electron g g 39 L 1000 energy des1rable 3 39139 l h 135 1sa2 220 249 I III I I Ill llil I I LI 1n 5390 100 150 200 mZ 250 400 220 1nformatlon at 3 WV h39 h mz 7 eg 300 1g g E 200 192 249 A Yes E 100 103 V 42 751 l 1535 B39 NO 56 100 150 200 mz 250 C Not sure Figure 13 Two spectra of the lactam illustrated Although the relative intensity of the molecular ion peak is greater at lower ioniza tion energy its absolute intensity as read from the left hand scale is actually somewhat reduced 25 E1 Fragm entat1on vs Molecular Structure IEEEEEEEEUUU From Lambert 100 152 MV39 100 8 8039 i 80 392 3 6039 60 CH CH3 lt 40 40 E g 20 20 l 152 M 1 r 1 l 391 u 7 l i 0 60 140 180 i0 0 13900 13940 180 mz Figure 134 Contrasting degrees of fragmentation observed in the El spectra of an aromatic and an alicyclic compound The former resists dissociation and gives a molecular ion from which the molec ular weight 152 Da is measured From PJ Ausloos C Clifton OV Fateev AA Levitsky SG Lias WG Mallard A Shamin and SE Stein NlSTEPANIH Mass Spectral Library Version 15 National Institute of Standards and Technology Gaithersburg MD 1996 26 Breakdown Curve for lpropanol CH3CHZCHZOH This information can be precisely determined using electrons of a single energy and scanning the energy This is what is different between molecules prev slide Relative Abundance me Lambert Clicker Q on Breakdown Curve Chemical ionization transfers a 00 50 3 very well de ned amount of energy to the analyte molecules Which of the following spectra are possible with Cl Relative Abundance 0 0 5 00 2 6 8 I 5 9V I 29 31 3 A All of them 29 B None of them 31 60 4 Confl ingjnly 2 39 D Only3 E l 2 and 3 22 Alto Breakdown Curve amp Internal Energy 0 Distribution 0 e eV of Molecular 90 so 51 w o m an Relative Abundance C hemical Ionization Ions I CE N2 Range c PK 42 eV me Lambert Breakdown Curve amp Internal Energy Distribution as me Ragga n of Molecular Ions II 6 421v l 3 We Ralanvn Abundance me Lambert g a a E z 5 39g 29 guvuMZE CamuanaHub39aakdmvnmrrveu nvanmlrmsvc lt i 5 mm W m My mm mm m mnmm immanan Lethmnues undev parlmu nr exucrimama und 2 an m mm mm mm of mm a Wm m 3 59 2 4 s 5 9V Breakdown l curve Fr El LowEnergyD CE N2 Range 9 E E e 1 l E Internal 2w 5ev 42w 6 E 5 Energy A l A J e 2 3 D t b t 7 3 IS r1 u 10n g 5950 g 3 50 g al a u u 1 1 g g 59 z of Molec g g a q a m 29 z 3 a 3 5 PM Ions III 5 5 g 5 mm 2 quotV1 at quotV2 E quotquot2 Reprodu01b111ty 4 Spectra of 1Pr0pan01 1n NIST mu Cnnm39hmm ms39r W 3 Mms Emma Cnntrihlltnrmlsnfrnm Dam 2mm 19m 5139 5D w W Vuu 29 42 59 2 2 u 5 III L s 5 III III II mamhbM 7mm WWW WW Cnntrihlltnnchanical mo Cn m39 t Cnnczpts Japan AISTNIMC Dahlia 5 mm 55 DH 29 42 59 27 42 I Iu In I LI Il w 57 3 40 5 an m 339 41 539s 5 7 u m 2 u 239s vephb 1Vmpana Mm 1Vmpana Electron Ionization Notes IllllllllElEl Big advantage high reproducibility of the fragmentation because Purely physical not chemical process Fragmentation involves only gasphase unimolecular reactions However all MS are far less reproducible than those based in the interaction of electromagnetic radiation with matter IR NMR MS depend on distribution of electron energies time allowed for fragmentation Advantages Disadvantages of El lllllllElElD TABLE 136 Advantages and Disadvantages of El Consequence Advantage Reproducible method Libraries of El spectra allow compound identification Extensive fragmentation Molecular structure information can be deduced occurs Ionization efficiency high Method is sensitive 1 in 1000 molecules is ionized Ionization is nonselective All vaporized molecules can be ionized Disadvantage Only positive ions formed Not ideal for some classes of compounds Radical cations formed Rearrangement processes complicate mass spectra Sample must be volatile Limited to low molecular weight compounds of approximately 600 Da or less Ionization is nonselective All vaporized molecules contribute to the mass spectrum Relatively energetic large Often extensive fragmentation limits value in molecular internal energy method weight determination From Lambert IIIIIIZIZIEIEIIJ Appendix Filament Emission and Failure Characteristics 35 Filament Emission Characteristics 1 IIIIIIZIZIEIEIIJ Filament blows 3 E quot6 gt 5 2 mA emission a 239 or S is gt Emiss1on begins E q 5 a 1 LTquot The Mass Spec Filament begins to glow Handbook of Service Published by Scienti c Instrument Services Inc http WWW sisweb com 0 I I I l l 0 1 2 3 4 5 Filament Current in Amps 36 Filament Emission Characteristics II II IIIIIIIIIIIIEIEIIJ 5 I m Filament blows 8 4 E 9 S S 3 E 9 5 O 2 c 9 U 9 E LL 1 Filament begins to glow 0 MS Handbook 0 i 392 393 394 jg Of Service Filament Current in Amps 37 Filament Burnout Patterns Filament Burnout Patterns The three pictures on this page show the filament burnout patterns of filaments studied in the above experiments As can be seen in Figure 1613 below when a filament burns out quickly by passing exces sive current through a new filament upon initial installation the filament breaks cleanly and evenly If the two filament halves were pushed together the breakpattems would match The gap between the two filament halves is due to the filament tension applied when the filament was installed on the source block and is not due to loss of the rhenium wire In some cases particularly in ribbon type filaments the ends of the filament will have slightly melted and fused The breaks will occur in the middle of the filament II IIIIIIIIIIZIEIEIIJ Figure 16 13 HPMSD filament burnout due to excessive current where the temperature is the hottest The pictures in Figure 1614 and Figure 1615 are very similar Figure 16 14 HP MSD filament norma burnout after 30 hours operation at 5mA emission current MS Handbook Of Service Figure 16 15 HPMSD filament normal burnout after 300 hours operation at 05mA emission current 38 Review of Fundamentals CHEM5181 Fall 2007 Prof Jose L Jimenez Business Items Fundamentals review used to be a homework trying clickers this year Homework assigned later today Fundamemab Partially based on clicker responses TOFMS Precision vs Accuracy Measure two variables Eg concentration of Na and CI in seawater Accepted value at origin Precision vs Accuracy C CQ Which is the most Qrecise A B C D I don t know Precision vs Accuracy III B C CQ Which is the most accurate A B C D I don t know Follow up Which is better A or B Precision vs Accuracy IV Precision Agreement between two or more measurements made in an identical fashion Accuracy Accuracy is the nearness of a measurement to the accepted value CQ This measurement is A Accurate B Precise C Precise and Accurate D Neither E I don t know Clicker Q Random errors are often distributed according to the normal error law Normal Probability Density Function CQ the probability that the absorbance is exactly 049 is A 049 B 25 C in nity D zero E I don t know 051 047 049 Absorbance unitless CLT httpwww chem um xDolets enfmll imit Anni entran imit htmi PDF vs CDF Probability Density vs Cumulative Probability Normal Probability Density Function Normal Cumulative Probability Function 1 2 Cumulative Probability E E E n El 051 045 D46 as am 047 049 047 Ma Ma Absorbance unitless Absorbance unitless CQ the probability that the absorbance is between 048 and 049 is A zero B It is not de ned C 037 D 056 E I don t know gt mUOw Fourier Analysis H3 is the Fourier M transform of h1 H3 is the FT of h2 H4 is the FT of h1 B and C are true All are false mm VWWV chem uua grApplEtsAppletFuurAnalAppLFuurAnalZ htrnl Clicker Q What is the strength of the electric field between two parallel plates at 1000 and 1000 V with respect to ground if they are separated by 5 mm 2000 Tesla 400000 Voltsmeter Zero 400000 NewtonsCoulomb 2000 Coulombs mpomgt Clicker Q What is the strength of the electric field between two parallel plates at 2000 and 4000 V with respect to ground if they are separated by 5 mm A 2000 Tesla B 400000 Voltsmeter C Zero D 400000 NewtonsCoulomb E 2000 Coulombs Clicker Q What is the net electric force on a neutral mercury atom in an electric field of 106 Vm It depends on the polarizability of Hg 16 X 1013 Nevvtons 16 X 1013 Nevvtons meter 106 Nevvtons Zero mpowgt Clicker Q What the force acting on a singly charged potassium ion with zero velocity in a 1 Tesla magnetic field 1 Newton 16 X 1019 Nevvtons Zero It depends on the relative alignment 1 Tesla moowgt Clicker Q What is true for the distribution of molecular speeds of N2 at ambient T amp P The average speed is 472 ms The minimum speed is 0 ms The maximum speed is not bound None of the above A B and C moopagt Clicker Q The average thermal speed of SF6 at ambient T amp P is Smaller than that of N2 Largerthan that of N2 Same as that of N2 None of the above A B and C ITIUOUJZD Clicker Q Air molecules in this classroom are colliding with each other approximately Once a second 100 times per second 10000 times persecond 106 times per second 1010times persecond ITIUOUJ Clicker Q The time between collisions for air molecules in an ionizer at a pressure of 10394 Torr is approximately 010003 01ns lus 100us 10 ms 3s Clicker Q What is the condition for laminar flow in a tube A Pressure lt 1 atmosphere B Reynolds number lt 1 C D E Reynolds number lt 2000 39 T lt Tcritical and PltPcritical What is laminar flow Clicker Q What is the vapor pressure ofwater at 100 degrees C A 1012 Torr B 760 mbar C 1 bar D 1 atmosphere E 15 Torr Linear Regression Standard regression l minimizes sum of squared residuals Residual vertical distance between datapoint and line Depending how much scatter there is in the data the slope and intercept will have more or less error ymism Xbisb Not displayed in simple regression in Excel Only gives y m x b Hr Need to used advanced red The Trouble w Standard Regression Every point pulls the line towards itself With a weight equal to the squared residual Noisy points outliers can seriously distort fit More Complete Regression Nonparametric regression Does not assume a distribution Typical linear regression assumes no errors on X Gaussian errors on Y More robust in the presence of outliers L L 39 Theil2htm Regression with errors in X and Y Weighted linear regression Different points have more or less error Numerical recipes for explanations Chapters 14 amp 15 httpwwwnrcom Different regressions in many progra Lecture Cl Part 1 Introduction and Theory of Chromatography CU Boulder CHEM 5181 Mass Spectrometry amp Chromatography Prof Jose L Jimenez Fall 2007 Reading Braithwaite amp Smith Chapters I amp 2 gggg rm m W 1 mm Chromatography is a physical method of separation in which the components to be separated are distributed between two phases one of which is stationary stationary phase SP While the other moves mobile phase MP or eluant in a de nite direction smmmm n l yr Why is Chromatography So Successful IIIIIIIIIIEIEI Tlma I mlnl Thermogram m w 15 2a 25 an as 4 l I r 1 1 9 I I I I I I I 1 I n r 1 n n I u I r I I 39 mast 4 ml 155 Ziemcmn eta an 39 J UC Riverside EDI r 739 m A m a9 A 2 f 39 20 a s alIIII39IILIIJ IT IIIIII39 I I39 39lII 2D 25 31 3935 41 d5 Sill Ha panzm Tkampaamtum DC1n Tima 1min 1 15 to 2 5 3D 35 40 IE 5n 3 E E a E 395 1 E E Chromatogram w Vapnmnr Temperature WC 5 l A A A A l A A A A l A A A A l A A A A l A A A A l A A A A l A A A A l A A A A l 0 5 10 15 I 15 30 35 40 Schematic of Column Chromatography Data to Computer Mobile Phase Sequence of events At t0 we will open the gate and let the analyte into the column Analyte will be carried by mobile phase Analyte may partition to stationary phase Analyte will be detected by its absorption of light at the detector Schematic of Column Chromatography 11 l I V V V I V V V I V V V I V V V l For simplicity we will assume that the mobile phase moves in steps rather than continuously If analyte had no affinity to the stationary phase it would just follow the mobile phase 7 Emerge at the detector after tM mobile phase time Schematic of Column Chromatography III A A A A AAA AAA AA AA AAA AAA AAA 0 If analyte has some affinity to the stationary phase it will be retarded Equilibrium K i Kinetics M Molecular mass transfer diffusion Emerge at the detector after retention time tR Retention and Mobile Phase Time IIIIIIIIIIIIIEIEID D g Analyte 0 peak I M 8 lt gt 0 B Unretamed 8 peak Simplest chromatogram W 2 components Unretained peak tM Analyte peak tR Corrected retention time t R 9 Chromatography Simulator Fraction per Theoretical Plate Inves ate a r Effect of K it If Effect of N 3 it iquot Effect of RR Enl utes Signal vs Ti me Distribution Relative ratio rearrange W 39 i Detector signal D II l u r Fll Went plates 1 U D I l Hater D I U CI 00 Hanna httpWWWchemuoagrAppletsAppletChromApplChrom2html Equilibrium the Distribution Ratio Stationary phase mobile phase amp analyte form a ternary system Each analyte is distributed between the two phases in equilibrium 7 Distribution ratio 7 CS concentration of analyte on the stationary phase 7 CM concentration of analyte on the mobile phase Using the Distribution Ratio Single Step Plate A Analyte A Q Analyte B Q If each symbol represents a umol given 10 cm2 of surface to which analytes adsorb and 100 cm3of liquid in which analytes are dissolved what are KA amp KB Large K has more af nity for stationary phase Small K has more af nitiy for the mobile phase Stationary Phase The rst step ina separation IIIIIIIIIIIIIIEIEIIJ Q does molecularlevel kinetics matter Key concepts Enriched in component which prefers mobile phase Not very good separation in lstep like thermogram It is repetition that makes it great Mechanisms of Partitioning to the Stationary Phase IIIIIIIIIIIIIIIIEIEIIJ Dissolve into the bulk Stick to the surface Q A Left is Absorption Right is Adsorption B Left is Adsorption Right is Absorption Separation Mechanism IEIZIEEIIZIEIEIIJ Direction ofmnbila phneflow Dntactor Chromatogram In MS we used hquot handles provided by Electric and magnetic Man of solute in mobile pha A A forces on ions In Chrom the handles v Concentration of salute in mtiomry Dhli are provided by component af nity for the SP and MP exceptions V Need to consider 0 Equilibrium mam m D Kinetics hs Fraction of bed length Partitioning in Real I Chromatography IIIIIZIEIEIIJ Mean Mobile phase I I l q quot 12 quotquot 7 Stationary phase Equilibrium distribution of molecules I l Actual non equilibrium distribution Figure 25 Equilibrium process during separation Factors In uencing Retention are those that in uence distribution equil Stationary phase type amp properties Mobile phase composition amp properties Intermolecular forces between Analyte amp mobile phase Analyte amp stationary phase Temperature Clicker Question A The chemical nature of the mobile phase affects distribution in gaschromatography GC amp liquid chromatography LC B The mobile phase affects distribution in GC but not LC C The mobile phase affects distribution in LC but not GC D The mobile phase affects doesn t affect distribution in either GC or LC E I don t know Intermolecular Forces I IIIIIIEIIJ Based on electrostatic forces Likeattracts like or oil and water similar electrostatic properties Polarpolar amp nonpolarnonpolar Molecules with dissimilar properties are not attracted Polar retention forces Ionic interactions IC Hydrogen bonding permanent dipoles DipoleInduced dipole component molecule 6 51gt stationary phase l9 Intermolecular Forces II I39Dipole Polar forces cont Energy of dipoledipole interaction IIIIIIIIIIEII Table 21 Dipole moments of some organic groups debye units R CHZCHZ RmO Me RNH ROH R COOH RCl R COOMe R CHO R CO R RCN 2 i l g D r6ij u dipole moment A analyte S stationary phase Alkene Methyl ether Amine Alkanol Carboxylic acid Chloride Methyl ester Aldehyde Ketone Nitrile UA 04 13 14 17 17 18 19 25 27 36 Factor of 10 variation on permanent dipole moment Factor of 104 variation on interaction energies As r6 gt mainly at the surfaces 20 Intermolecular Forces Ill London IIIIIIIIIIIIIEIIJ London s Dispersion Forces Most universal interaction between molecules Only one for nonpolar species Due to induced dipoles Relatively weak Energy of interaction 30A05PIAIS 8L 6 2r IA 15 a is the polarizability I ionization potential A analyte S stationary phase 21 I Separating Ef ciency I Peak Width IIZIEIZIIZIIZIEIEID 1214h Tangents drawn a Assume Gaussian Peaks to the inflection points h FWHM wh 2360 HWHM 1180 Base Width of Peak wk Width at intersection of lt 0607h 20 tangents at in eXion Ingifglon points a and the P ash 23550 baseline wb 4 0 955 of molecules are Within wb 1 4h 4 Assuming that the 3 0 3 0 Gaussian model holds When in doubt use NORMDIST in Excel Peak heights of a Gaussian peak and width as a function of standard deviation I Figure 23 Braithwaite amp Smith I 22 Resolution in Chromatogtdglgan Objective accurate measurement R At of individual peak areas spectra 5 2 FWHM E 25 2 2 39 What is the g resolution this is a E 15 a mass spectrum g 1 39 If it is a g ehromatogram E 05 11 0 Clicker Question Analysis B is more desirable than A A In MS amp ChIorn B In MS but not Chrom C In ChIorn but not MS D In neither MS nor Chrom E lneed a coffee Resolution vs Peak Integration EEEEEUUI i39 Overlapping Peaks Peak Fosilltm Peak Width Peak Height Li J E Ll l H j J Li 1 Automatic Abw ILEN Area ul Blue Peak This Sllm height it i httpWWWViasorgsimulationssimusoft peakoverlap html Can One Have Too Much Resolution EEUUI R8 08 i Poor resolution Good resolution adequate selectivity and selectivity but poor ef ciency too long analysis time resulting in poor ef ciency and band broadening Excessive Good ef ciency adequate resolution selectivity satisfactory excellent analysistime selectivity and ef ciency analysis time toolong 12ltRS lt15 Figure 28 Resolution selectivity and column el ciency Progress of a Chromatographic Analysis EEEDDE 0 If you have too much resolution you can Shorten the column Increase temperature GC ow rate gt Shorten the analysis M M Distance along column 27 Concentration in stationary phase Issues for Start of Class IIIIIIIIIIIEIEIEI For review lecture clicker question send 3 slides about instrumentation interpretation chromatography CP Return clickers at end of review lecture 0 No additional HW Will distribute past HWs FCQ evaluations This year they are online for the first time Do separately for Joel and Jose 0 Zoomerangcom questionnaire Will send invitation by email done on web Typically more detailed and useful Anonymous Class participation points to everyone for each response Lecture C2 Electric FieldDriven Separations Ion Mobility Spectrometry Gel Electrophoresis and Capillary Electrophoresis CU Boulder CHEM 5181 Mass Spectrometry amp Chromatography Prof JoseLuis Jimenez Fall 2007 R Weinberger Practical Capillary Electrophoresis Academic Press 1993 Objective of Today s Lecture Learn about separations driven by electric elds Not under vacuum unlike MS In a gas Ion Mobility Spectrometry In a liquid Electrophoresis We teach them under chromatography but chromatographic principles partitioning between phases do not apply in most cases Books on Electroseparations Author Publisher Title A3211quot Comments Rubinson amp Prentice 39V quot quotquot Good Basic Chapter ofCE 2000 127 Rub39 5 Haquot Ion q 5 Rm 7000 In Library d 39Ms Good reference with lots of Weston amp HPLC amp CE 1997 Brown Academic 00790454 H63 82 pointerstzrtal research Good practical reference Curtlco I Bay I Basic HPLC and CE of Blomolecules read it ifyou use these Goodlng amp Bloanalytlc 199 40 techniques a lot for Wehr a Labs QP5199H53 C86 iomo ecu es Practical Capillary Electrophoresis 2quot Weinberger Acgliirglc Ed 2000 105 General reference on CE QP5199C36 W45 2000 Part 1 Ion Mobility Spectrometry llass spectrometry without the vacuum Electrophoresis in the gasphase A Common Application of IMS A Common Application of IMS Ion Mobility Spectrometry I 0 OJ O 3m 30 ado Of 1 4 O 1 Electric force pulls Drag of gas retards Ratio is Ion mobility LAN L distance Questions 0 How many collisions does an ion experience every ms 0 Do the ions continuously accelerate Ion Mobility vKE dKlE Where d is the distance traveled cm K is the ion mobility cm2 V391 s39l 7 Increases with number of charges 7 Decreases with cross sectional area bigger target tis the time s E is the electric field strength V cm39l Ifdis fixed and K1 gtK2 Dri H gas Co rl39mr gas l39araday plate Sllullvr grid Ion Mobility Spectrometry H Membrane W n gt ha inple of lnlei 4 Idea race of ions most mobile get to detector rst 3000 V Does this remind OH 0 FIGURE 1617 A y f Illustration of the construdjon of an ion mobility spectrometer with a something membrane input An example or a membrane is a 20 lm39l hick dimeliiylsiiicone sheet The organic analytes are soluble in h L 39 m l but a ma lo a high degree The shutter er gate grid is an array at line wires Spaced abouti mm apart lliat stops ions from traveling larmer down the drift tube It is pulsed open lor times in the range 0 054 ms Typical values are 25 ms pulse lime and 25 ms drill lime The pulse L e ZDHBV zones furthen Reprinted With pelmissinn rrem Analytical Chemistry Copyright 1991 American V y Chemical Soclet J From Rubinson Example of Ion Mobility Spectrum IIIIIIIIIIIEI b iReactant ions Drift timems FIGURE 1616 A Ion mobility spectra of volatiles from wood a Negative ion mobility spectrum of jack pine heartwood b Positive ion mobility spectrum of balsam fir heartwood The samples were a few particles 100 200 ug placed in a 10 cm X 3 mm glass tube with a nonreactive plug in it to prevent solid particles from entering the spectrometer The volatiles were carried into the spectrometer by a stream of purified air Refz Redrawn with permission from Lawrence A H et al 1991 Analytical Chemistry 63 1217 1221 Copyright 1991 American Chemical Society From 1 H150 Pros and Cons of I I II lllllll Advantages Very fast Continuous Low detection limits ppb Relatively cheap Disadvantages Low resolution Potential interferences 0 Chemical ionization reactions gt Matrix Effects 0 Clustering of ions AHZO2 AHZO3 AH24 Part 2 Gel Electrophoresis Electrophoresis I Electric force pulls Drag of gas retards Ratio is Ion mobility distance Concept of Electrophoresis 0 Used mostly to separate charged molecules 0 Based on differences on molecular movement through a uid carrier electrolyte or buffer under an electric field 0 No partitioning between mobile and stationary phases Not a chromatographic technique Result called an electropherogram Physical Basis of Electrophoresis 0 Solutions can be electrically conductive Due to the migration of individual ions 0 Different ions migrate at different rates Electrophoresis means ion migration Analytical EP uses the differential ion migration rates as a means of separation Electrophoresis vs Chromatography Table 41 Comparison of Electrophoreuc and Chromatographic Tevms Capillary ulcclmphomsis Ihronmlog iaph Elemmphemgmm Chrom lugmm Applied potential Flow rate 39an icr ulcclmlyw or boner Elucm or mobile phmt Injection mode hydrostatic or cleclmmlgrmion Injector Mlkmli 39 Relemm Iimc Llccucphorcuc mobility Column 01pmin actor Velour 7 Eleclroosmotic m Highrvolmge power supply Pump apillnry Column De nition of Ionic Mobility in Solution Fmg v v relative Velocity lt between the ion and F212 the uid Ionic Mobility F mg 67er F 212 4E Units cIn2 s
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