hmotnostních spekter 1

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1 Klepnutím lze upravit styl předlohy podnadpisů. Projekt vzdělávání Ekotech 2012 Základy interpretace hmotnostních spekter 1 Petr ŠIMEK 1

2 Practical approach Mass Spectrometry Obecné principy i a základy 2

3 Flow Chart of the Instrumental Analysis Analytical problem Sampling Sample preparation Instrumental analysis Separation Detection Chromatography/Electrophoresis Spectroscopy Data acquisition/processing INFORMATION Management 3

4 MASS SPECTROMETRY (MS) Physical method of IONIZATION, SEPARATION and DETECTION of molecules according to their MASS m a CHARGE z. i.e. m/z effective mass m ( 12 C) = 1.66 x kg z (electron) = x C Energy = 100 kj/mol = 1.04 ev = 23.9 kcal/mol 1 mol = molecules Hypotetický nositelhmotnosti Higgsův boson 4

5 The Periodic Table Groups All Elements, periodická t. Chemical symbol Mass Atomic number = number of electrons and protons Elements in columns have similar chemical properties p Note biggest mass! Number of neutrons = mass - Z Common organic element Often encountered in MS Important trace element 5

6 kg yoctogram (yg) Praktická MS a separace malých molekul Mass Scale Orders of Magnitude Electron, the lightest elementary particle with a measured nonzero rest mass kg Atomic mass unit (amu) or dalton (Da) kg Proton (938.3 MeV/c²) kg Hydrogen atom,, the lightest atom kg Neutron (939.6 MeV/c²) kg Water molecule ( amu) kg Z boson (91.2 GeV/c²) kg Top quark (173 GeV/c²), the heaviest known elementary particle kg Caffeine molecule (194 amu) kg Lead-208 atom, the heaviest stable isotope known zeptogram (zg) kg Haemoglobin A molecule attogram (ag) 6

7 Mass Spectrometry Basic Prerequisites základní podmínky Molecules of the analyte must get an ELECTRIC CHARGE! Elektrický náboj Separation of ions in an analyzer requires high vacuum! min 10 3 Pa (10 5 Vakuum ) torr sufficient for mean free path of 1m For pumping by rotary pumps, high vacuum delivered by turbomolecular pumps 7

8 Mass Spectrometry Formation of ions is a chemical reaction! Destructive method with minimal sample consumption Separation method History (historie) : first applied to separation of isotopes (Thompson, 1913) after world war II since 60th developement in petroleum industry GC MS since 1976 first LC MS 1984 Fenn et al electrospray 1989 Hillenkamp, Karas MALDI 2002 Fenn, Tanaka Nobel price for protein MS 8

9 Appearance of the Mass Spectrum Y axis Rl Relative intenzityi X axis Effective mass m/z Relative Abundance of the Ions in the Spectrum Depends on their Stability!!! Relativní výskyt iontů v záznamu spektra závisí na jejich stabilitě!!! 9

10 Element Rel. Atomic Mass Isotopic Composition Standard MW Type 1H (4) (70) (7) 2D (4) (70) M+1 4He (10) (3) 12C (0) 98.93(8) (8) 13C (10) 1.07(8) M+1 14N (9) (7) (2) 15N (9) 0.368(7) M+1 16O (15) (16) (3) 18O (9) 0.205(14) M+2 19F (7) (5) 23Na (23) (2) 31P (20) (2) 32S (12) 94.93(31) (5) 33S (12) 0.76(2) 34S (11) 4.29(28) M+2 35Cl (4) 75.78(4) (2) 37Cl (5) 24.22(4) M+2 39K (3) (44) (1) 40K (3) (44) (1) 41K (28) (44) M+2 79Br (20) 50.69(7) (1) 81Br (3) 49.31(7) M+2

11 Isotopic Abundance 11

12 Elements and Isotopes, prvky a izotopy Chemically pure analyte is a mixture of isotopes Common net elements H(D), F, P, I (only a single isotope) M+1 elements 12 C/ 13 C, 14 N/ 15 N, M+2 elements 16 O/ 18 O, 28Si/30Si (100:3.4) 32 S/ 34 S (100:4.4) 4) 35 Cl/ 37 Cl (3:1), 79 Br/ 81 Br (1:1) Combination of elements their distribution is binomic (a+b) n e.g. for Br 2 1:2:1 (a 2 +2ab+b 2 ), for Cl 2 9:6:1(9a 2 +6ab+3b 2 ) etc. 12

13 Elements and Isotopes If M2 M+2 < 3% M2 M+2 elements Si,S,Cl,Br S not present Characteristic combinations of isotopic abundances 13

14 Elements and Isotopes M+1 elements 13 C/ 12 C = 1.1% net signal intensity is divided by N/ 14 N = 0.37% net signal intensity is divided by

15 Rings plus Double Bonds Rule (určení počtucyklů a násobnýchvazeb) rdb rule Total number of rings and double bonds in a molecule C x H y N z O n R+db = x ½ y + ½ z + 1 C Si, H X, N. P, O...S Values 0, 1, 2, 3, 4. Odd electron ion (OE)+. Vl Values ½, 1 ½, 2 ½, 3 ½ Even electron ion (EE)+ Consequence: EE ion cannot be cation radical ion M +. C 2 H 5 OH * ½ + ½ * = 0 OE C 4 H 11 N +. 4 ½ * 11 + ½ * = 0 OE C 6 H 5 CO + 7 ½ * 5 + ½ * = 5.5 EE 15

16 Ringsplusdoublebonds bonds rdb rule Napthalene C 10 H 8 5 double bonds + 2 cycles 16

17 Dusíkové pravidlo Praktická MS a separace malých molekul The Nitrogen Rule Nitrogen the only element with even mass and odd valence If a compound contains odd number of nitrogen atoms, its molecular ion will be at odd mass number. If the compound does not contain nitrogen or has even number of nitrogens, its molecular l ion is at even mass number. Lichá hmotnost = lichý počet atomů dusíku v molekule 17

18 Ions A charged form of an atom or molecule Formed by adding or subtracting a charged entity ( ionization ): M e > > M +. Kation radikál M + H + > MH + Kation Simplest ion is H + which is equivalent to a proton Electron ionization Chemical ionization Ions are fundamental to mass spectrometry: they can be manipulated with electric and/or magnetic fields. Neutral molecules cannot MS measures m/z (mass divided by charge), so is the same as

19 Appearance of the Mass Spectrum Y axis Relative intenzity X axis Effective mass m/z 19

20 Classification of Ions Klasifikace iontů OE +. ions reactive, not stable, rare in low mass region lichý e most typical decomposition to EE+ and R. Odd electron ion OE +. EE + + R. OE +. ion is formed from M +. by cleavage of two bonds (typical for reactions and rearrangements of a ring) EE + ions generally more stable, fully paired electrons in sudý e outer shell Even electron ion EE + The ease of their ionization n > π > σ MH + is another EE + ion (typical ldecomposition i to another EE+ and N (neutral fragment) OE +. EE + + N 20

21 WhichAnalytes Are Easily Ionized? That possessing : non/bond n electrons (nevazebný &volný elektronový pár) heteroatomy N,O,S, halogens > π conjugated electrons, aromates > π nonconjugated electrons (alkenes) > σ electrones (alkanes, C C bond) > σ electrones (alkanes, C H bond) 21

22 MASS (Hmotnost) Mass Unit (u) is also called Dalton (Da) All mass spectrometers measure m/z and never mass. Monoisotopic i Molecular l Wih Weight Weight of predominant (non average) isotopes. eg. : Stearic AidCH Acid 3 (CH 2 ) 16 CO 2 H is Da Average Molecular Weight Average weight of all Isotopes. eg. : Stearic Acid CH 3 (CH 2 ) 16 CO 2 H is Da 22

23 MASS NAPHTHALENE C 10 H (mainlib) Naphthalene Monoisotopic Molecular Weight Average Molecular Weight

24 MASS LEUCINE C 5 H 5 N 1 O OH 50 O 30 NH (mainlib) Leucine Monoisotopic i Molecular l Weight Average Molecular Weight

25 MASS RESOLUTION Mass Resolution (rozlišení) The ability of distinguish two different masses Eg: Da vs Da R=m/Δm Mass Accuracy (přesnost změřené hmotnosti) The ability to obtain the exact or true mass of a compound unit millidalton percentage ppm (parts per million) 25

26 MASS RESOLUTION Mass Resolution/Mass Accuracy Mass resolution: represents the ability to separate two adjacent masses. It measures the "sharpness" of the MS peak. Mass accuracy: indicates the accuracy of the mass information provided d by the mass spectrometer. 26

27 MASS RESOLUTION Resolution calculation: R=m/Δm 27

28 MASS RESOLUTION Nominal unit resolution High resolution 28

29 MASS RESOLUTION High resolution Nominal resolution (R= 6000) (R=1000) 29

30 What type of information can be obtained by Mass Spectrometry? Qualitative i Molecular weight Elemental Composition Structural Information Chromatographic Peak Purity Quantitative Trace Level Analyses 30

31 Componentsof of a Mass Spectrometer Ionization/ desorption Mass Sorting Detection + Source Form ions (charged molecules) Analyzer Sort Ions by Weight Ion Detection Detect t ions 100 Inlet Solid Liquid Vapor Sample Introduction Method to vaporize sample Data Analysis 31

32 Current Mass Spectrometric Working Range Amount [Moles] Number of Molecules Weight Peptide Reserpine Peptide Reserpine [grams] ng 1 pmol 1.64 pmol 6.02 x pg 1 fmol 1.64 fmol 6.02 x fg 10 attomol 16.4 attomol 6.02 x fg 1 attomol 1.64 attomol 6.02 x ag zeptomol x zeptomol 1 mol = mol. weight x gram 1 nmol = mol. weight x nanogram 1 pmol = mol. weight x pikogram 1 fmol = mol. weight x femtogram 32

33 Ionization Techniques Electron Impact (EI) Chemical Ionization (CI) Fast Atom Bombardment (FAB) API MALDI Hard, Fragments Soft, Intact 33

34 ELECTRON IONIZATION e - beam Lenses Heated filament Analyzer Vacuum system (10-5 to 10-6 Torr) Ionization by electron beam M + e > M e Very energetic; M +. will typically fragment Fragments can distinguish structure Must be able to get sample vapour into source Goodfor thermally stable analytes with low polarity 34

35 Chemical Ionization 1 Torr reagent gas, eg e.g. CH 4 e - beam Heated filament Analyzer Vacuum system (10-5 to 10-6 Torr) Reagent gas ionized by electron beam CH 4 + e > CH e CH 4+ + CH 4 > CH 5+ + CH 3 CH + > 5+ + M > [M+H] + + CH 4 Mild; MH + doesn t fragment as much as with EI Good for thermally ysabea stable analytes ay with low polarity 35

36 APCI (Atmospheric Pressure Ionization) ionizace za atmosférického tlaku Heated Nebulizer: Atmospheric Pressure Chemical Ionisation (APCI) corona discharge needle polar to unpolar and thermally stable compounds 36

37 APCI Heated Nebulizer: Atmospheric Pressure Chemical Ionisation (APCI) corona discharge needle polar to unpolar and thermally stable compounds 37

41 APCI Atmospheric Pressure Chemical Ionization Features: Entire mobile phase and sample vaporized into the gas phase (with heat), then ionized Accommodates high LC flow from (0.1-2 ml/min) Uses heat ( C) & nebulizer gas to vaporize HPLC eluent and steam distill sample into the gas phase for APCI Temperature setting is not critical Detection limits are very good Limitations: it ti Thermally labile analytes may degrade when vaporized 41

42 APCI Inlet Suitable for polar, thermally stable compounds Usually, Molecular Weight < 1000 amu Probe is heated to facilitate vaporization Requires nebulizer and auxiliary gas Requires corona discharge needle to produce ionization i (APCI) 42

43 Electrospray ionization - ESI Electric field on the electrospray needle 43

44 Electrospray ionization - ESI Droplet desintegration in time N R number of droplets droplet diameter [um] Δt evaporation time [μs] 44

45 Features: Praktická MS a separace malých molekul ESI Hot drying gas improves evaporation ofcharged droplets no thermal degradation better sensitivity at higher flow rates tolerates higher aqueous solvent compositions Improved performance over a wide range of LC flow rates without splitting (5µL/min to 1mL/min) Detection limits are very good 45

46 Application range of ESI, APCI and PI ESI 46

47 MALDI ionizacei Matrix Assisted Laser Desorption Ionization Desorpce analytů laserem v matrici 47

48 MALDI: Matrix Assisted Laser Desorption Ionization Sample plate hν Laser 1. Sample (A) is mixed with excess matrix (M) and dried on a MALDI plate. 2. Laser flash ionizes matrix molecules. 3. Sample molecules are ionized by proton transfer from matrix: AH + MH + +A M+AH kv Variable Ground Grid Grid 48

49 MALDI Sample Preparation MALDI preparation protocol: Dried droplet preparation: matrix solution mixed with analyte solution on the metal target and dried Typical DHB dried d droplet preparation on a steel target Matrices are e.g. 25dihydroxy 2.5-dihydroxy benzoic acid (DHB) or 4-hydroxy-α-cyanocinnamic acid (CHCA) Beavis, Chaudhary, Chait, Org. Mass Spectrom. 1992, 27, 156. Strupat, Karas, Hillenkamp, 1991, 111,

50 Desorption: Plume (chochol) Desorpce analytů v mikrosekundové sekvenci LIF images of the expansion of both light matrix (3-HPA) and heavy bimolecule (dye-tagged DNase I protein, narrow jet) during the MALDI process Puretzky, Geohegan, Chem. Phys. Lett. 1998, 286,

51 Matrix Selection Látky vhodné do matrice pro laserovou desorpci α-cyano-4-hydroxy-cinnamic y y y acid (CHCA) Sinapinic Acid 2,5-Dihydroxybenzoic acid (DHB) Super DHB 3-Hydroxypicolinic acid Peptides<10kDa Proteins >10kDa Neutral Carbohydrates, Synthetic Polymers Proteins, Glycosylated proteins Oligonucleotides HABA Proteins, Oligosaccharides 51

52 Terčíky na vzorky pro MALDI Praktická MS a separace malých molekul MALDI Sample Plate Options 96 x2 Close External Calibration with hydrophobic surface Gold 100 well 400 well with hydrophobic surface 52

53 Unique MALDI Features Soft ionization analyze biomolecules intact Sample in solid state Ionization and transfer of analytes of the broad mass range analyze a wide variety of biomolecules (> 300 KDa) High ionization efficiency and thus sensitivity (low fmole) Fast 53

54 Ionization methods : ESI vs. MALDI MALDI vers. ESI porovnání ESI Advantages Direct coupling of LC to MS Fast lots of MS and MS/MS Accepted MS/MS ionization mode ESI LIMITATIONS Limited time for MS/MS (limited depth of analysis) Miss high h MW peptides more difficult than MALDI? MALDI Advantages Sample in solid state Not time limited for MS or MS/MS Analysis can be faster or slower than separation Sophisticated workflows Fast lots of MS and MS/MS MALDI LIMITATIONS Offline coupling of LC? Miss low MW peptides Conv. MALDI not ideal for ID of unknown proteins 54

55 Combined Desorption and Electrospray ionization= DESI Ambientní ionizační technika, kombinace elektrospreje a desorpce Cooks et al, 2004, Science 2004: 306, Nabité mikrokapky tvořené elektrosprejem z mobilní fáze slouží jako projektily pronikající do povrchové vrstvy, předávají náboj (heterogenous charge transfer mechanism) a desorbují vznikající ionty, které jsou přenášeny pomocí vnějšího el. pole (droplet pick up mechanism) do vstupního otvoru MS Není ítřeba žádná á matrice, ti matrici tiitvoří vzorek sám Komercializace, Prosolia Inc., 55

56 Desorption Electrospray ionization = DESI Velké molekuly mechanismus ionizace podobný ESI, vznikají mnohonásobně nabité ionty

57 Desorption Electrospray ionization = DESI Malé molekuly přenos náboje mezi molekulou analytu na povrchu vzorku a iontem rozpouštědla v (a) kapalné nebo (b) plynné fázi DESI MS prstu 50 min po orální aplikaci 10 mg antidepresiva/antialergika Claritin (účinná látka loratadine, MH+ = 383.3) Mobilní fáze: MeOH-voda

58 Malé molekuly Praktická MS a separace malých molekul Desorption Electrospray ionization = DESI DESI MS povrchu ibiškového květu MP = EtOH watert 50:50 Detekováno: směs esenciálních olejů carotenoids antioxidanty typu 4-hydroxy-3,5-di-tert-butyl-p-cresol 35di t t l (m/z MH + = 219) Ionizace přímo též v plynné fázi mezi analytem a iontem rozpouštědla nebo částic ve vzorku

59 DART = direct analysis in real time Ionizace analytů na povrchu vzorku excitovaným plynem (He, N2) Ambientní ionizace, na rozdíl od DESI nevyžaduje ESI sprejovanou kapalinu Cody BC, Laramee JA, US Patent 6,949,741; 27/09/2005, Komercializace Jeol, IonSense DART scheme

60 DART MS Direct analysis in real time Není třeba žádná příprava vzorku nebo separace Analyt je přímo ionizován na povrchu vzorku Kapalina nanášena na skleněnou tyčinku a ionizována Těkavá látka vnášená přímo do přiváděného ionizovaného plynu Na rozdíl od DESI nevznikají adukty s ionty kovů a ionizace probíhá bez rozpouštědel Desopce je omezena na malé molekuly s minimální těkavostí Chernetsova E, Morlock E, Mass Spectrom Rev 2011, 30, Výbušniny toxiny léčiva drogy polární i málo polární sloučeniny cukry Výbušniny, toxiny léčiva, drogy, polární i málo polární sloučeniny, cukry, potravinářské

61 Direct analysis in real time DART MS Není třeba žádná příprava vzorku nebo separace Přímý vstup vzorku z otevřeného ř laboratorního prostoru

62 Forenzní DART MS analýza 5 různých druhů inkoustu DART MS Jones R et al, J Forensic Sci 2006: 51, 915

63 Praktické náměty O OH? NH 2 Uvažte, jak probíhá ionizace vašich oblíbených analytů 63