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Introduction to Walk-Up Mass Spectrometry Jonathan A. Karty, Ph.D. September 27 & 29, 2010.

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Presentation on theme: "Introduction to Walk-Up Mass Spectrometry Jonathan A. Karty, Ph.D. September 27 & 29, 2010."— Presentation transcript:

1 Introduction to Walk-Up Mass Spectrometry Jonathan A. Karty, Ph.D. September 27 & 29, 2010

2 Topics Covered Molecular Weight and Isotope Distributions Molecular Weight and Isotope Distributions Accuracy and Resolution Accuracy and Resolution EI, ESI, and APCI ionization EI, ESI, and APCI ionization EI Fragmentation EI Fragmentation A Handful of MS Applications A Handful of MS Applications

3 Why Mass Spectrometry Information is composition-specific Information is composition-specific Very selective analytical technique Very selective analytical technique Most other spectroscopies can describe functionalities, but not chemical formulae Most other spectroscopies can describe functionalities, but not chemical formulae MS is VERY sensitive MS is VERY sensitive MSF personnel dilute NMR samples 1:500 MSF personnel dilute NMR samples 1:500 Picomole sensitivity is common in the MSF Picomole sensitivity is common in the MSF Mass spectrometers have become MUCH easier to use in the last 15 years Mass spectrometers have become MUCH easier to use in the last 15 years

4 Three Questions Did I make my compound? Did I make my compound? Molecular weight is an intrinsic property of a substance Molecular weight is an intrinsic property of a substance Did I make anything else? Did I make anything else? Mass spectrometry is readily coupled to chromatographic techniques Mass spectrometry is readily coupled to chromatographic techniques How much of it did I make? How much of it did I make? Response in the mass spectrometer is proportional to analyte concentration (R = [M]) Response in the mass spectrometer is proportional to analyte concentration (R = α[M]) Each compound has a unique response factor, Each compound has a unique response factor, α

5 Common MS Applications Reaction monitoring Reaction monitoring Crude reaction mixture MS Crude reaction mixture MS Stable isotope labeling Stable isotope labeling Stability studies Stability studies Quick product identification (TLC spot) Quick product identification (TLC spot) Confirmation of elemental composition Confirmation of elemental composition Much more precise then EA Much more precise then EA Selective detector for GC/HPLC Selective detector for GC/HPLC MS provides molecular weight information about each chromatographic peak MS provides molecular weight information about each chromatographic peak

6 Important Concepts to Remember Mass spectrometers analyze gas-phase ions, not neutral molecules Mass spectrometers analyze gas-phase ions, not neutral molecules Neutrals don’t respond to electric and magnetic fields Neutrals don’t respond to electric and magnetic fields If a molecule cannot ionize, MS cannot help If a molecule cannot ionize, MS cannot help MS is not a “magic bullet” technique MS is not a “magic bullet” technique MS can describe atomic composition of an ion MS can describe atomic composition of an ion Connectivity of the atoms is much more challenging Connectivity of the atoms is much more challenging Although MS requires a vacuum, it cannot be performed in a vacuum of information Although MS requires a vacuum, it cannot be performed in a vacuum of information Deriving useful information from MS data often requires some knowledge of the system under investigation Deriving useful information from MS data often requires some knowledge of the system under investigation

7 Resolution is the ability to separate ions of nearly equal mass/charge Resolution is the ability to separate ions of nearly equal mass/charge e.g. C 6 H 5 Cl and C 6 H m/z e.g. C 6 H 5 Cl and C 6 H m/z C 6 H 5 Cl = amu (all 12 C, 35 Cl, 1 H) C 6 H 5 Cl = amu (all 12 C, 35 Cl, 1 H) C 6 H 5 OF = amu (all 12 C, 16 O, 1 H, 19 F) C 6 H 5 OF = amu (all 12 C, 16 O, 1 H, 19 F) Resolving power >4700 required to resolve these two Resolving power >4700 required to resolve these two Two definitions Two definitions Resolution = Δm/m (0.024/ = or 2.2*10 -4 ) Resolution = Δm/m (0.024/ = or 2.2*10 -4 ) Resolving power = m/Δm (112.03/0.024 = 4668) What is Resolution?

8 Resolving Power Example RP= 3,000 RP= 5,000RP= 7,000 All resolving powers are FWHM C 6 H 5 OF C 6 H 5 Cl

9 Mass Accuracy MSF reports mass accuracy as a relative value MSF reports mass accuracy as a relative value ppm = parts per million (1 ppm = %) ppm = parts per million (1 ppm = %) 5 mass 300 = 300 * (5/10 6 ) = ± Da 5 mass 300 = 300 * (5/10 6 ) = ± Da High resolving power facilitates precise mass measurements High resolving power facilitates precise mass measurements Accurate mass spectrometry is used to confirm a molecular formula Accurate mass spectrometry is used to confirm a molecular formula Walk-up instruments in the MSF should be treated as “nominal mass” accuracy Walk-up instruments in the MSF should be treated as “nominal mass” accuracy +/ Da mass accuracy +/ Da mass accuracy

10 A Discussion of Molecular Ions

11 Molecular Weight Calculations Calculate molecular weights of expected components PRIOR to performing MS The molecular weight of a compound is computed by summing the masses of all atoms that comprise the compound. The molecular weight of a compound is computed by summing the masses of all atoms that comprise the compound. Morphine: C 17 H 19 NO 3 = (17) (19) (3) = Da Morphine: C 17 H 19 NO 3 = (17) (19) (3) = Da Yet is observed by EI-MS Yet is observed by EI-MS Molecular weight is calculated assuming a natural distribution of isotopes Molecular weight is calculated assuming a natural distribution of isotopes Molecular weights calculated with average masses for Br, Cl, and many metals will differ greatly from MS data Molecular weights calculated with average masses for Br, Cl, and many metals will differ greatly from MS data

12 Monoisotopic vs. Average Mass Most elements have a variety of isotopes Most elements have a variety of isotopes C  12 C is 98.9% abundant, 13 C is 1.1% abundant C  12 C is 98.9% abundant, 13 C is 1.1% abundant For C 20, 80% chance 13 C 0, 18% chance 13 C 1, 2% chance 13 C 2 For C 20, 80% chance 13 C 0, 18% chance 13 C 1, 2% chance 13 C 2 Sn has 7 naturally occurring >5% ab. Sn has 7 naturally occurring >5% ab. F, P, Na, Al, Co, I, Au have only 1 natural isotope F, P, Na, Al, Co, I, Au have only 1 natural isotope Mass spectrometers can resolve isotopic distributions Mass spectrometers can resolve isotopic distributions Monoisotopic masses must be considered Monoisotopic masses must be considered Monoisotopic masses are computed using the most abundant isotope of each element ( 12 C, 35 Cl, 79 Br, 58 Ni, 11 B, etc.) Monoisotopic masses are computed using the most abundant isotope of each element ( 12 C, 35 Cl, 79 Br, 58 Ni, 11 B, etc.) For morphine, monoisotopic mass = For morphine, monoisotopic mass = ( * 17) + ( * 19) ( * 3) ( * 17) + ( * 19) ( * 3)

13 Isotopic Envelopes Mass spectrometers measure ion populations Mass spectrometers measure ion populations 10 2 – 10 6 ions in MS peaks 10 2 – 10 6 ions in MS peaks Any single ion only has 1 isotopic composition Any single ion only has 1 isotopic composition The observed mass spectrum represents the sum of all those different compositions The observed mass spectrum represents the sum of all those different compositions “M+ peak” “M+1 peak” “M+2 peak”

14 C 17 H 19 NO 3 Mass Spectrum 13 C 0, 15 N 0 13 C 1 or 15 N 1 13 C 2 or 13 C N avg. mass

15 Isotopic Envelope Applications Isotopic envelopes can be used to preclude some elements from ionic compositions Isotopic envelopes can be used to preclude some elements from ionic compositions Lack of intense M+2 peak precludes Cl or Br Lack of intense M+2 peak precludes Cl or Br Many metals have unique isotopic signatures Many metals have unique isotopic signatures M+1/M+ ratio can be used to count carbons M+1/M+ ratio can be used to count carbons [(M+1)/M+]/0.011 ≈ # carbon atoms [(M+1)/M+]/0.011 ≈ # carbon atoms For morphine: (0.1901/1)/0.011 =  17 For morphine: (0.1901/1)/0.011 =  17 Isotope table can be found on NIST website Isotope table can be found on NIST website Link from MSF “Useful Information” page Link from MSF “Useful Information” page

16 A few isotope patterns C 2 H 3 Cl 3 trichloroethane C 12 H 27 SnBr tributyltin bromide C 83 H 122 N 24 O 19 A 14-mer peptide

17 Last Comments on Molecular Ions Be aware of ionization mechanism Be aware of ionization mechanism EI, LDI, and CI generate radical cations EI, LDI, and CI generate radical cations M + is an odd electron ion M + is an odd electron ion Nitrogen rule is normal Nitrogen rule is normal Odd molecular ion mass implies odd # of N atoms Odd molecular ion mass implies odd # of N atoms M + for morphine by EI is , odd # N (1) M + for morphine by EI is , odd # N (1) ESI, APCI, MALDI, and CI make cation adducts ESI, APCI, MALDI, and CI make cation adducts M+H and M+Na are even electron ions M+H and M+Na are even electron ions Nitrogen rule is inverted for these ions Nitrogen rule is inverted for these ions Even molecular ion mass implies odd # of N atoms Even molecular ion mass implies odd # of N atoms M+Na for morphine by ESI is , odd # N (1) M+Na for morphine by ESI is , odd # N (1) Metal atoms and pre-existing ions or radicals can override these rules Metal atoms and pre-existing ions or radicals can override these rules

18 Some useful software tools The “exact mass” feature in ChemDraw will give you a monoisotopic mass The “exact mass” feature in ChemDraw will give you a monoisotopic mass Not always correct for complex isotope patterns Not always correct for complex isotope patterns Two freeware apps are available from MSF website “Links” page Two freeware apps are available from MSF website “Links” page These can be used to predict the entire isotopic pattern as an exportable image These can be used to predict the entire isotopic pattern as an exportable image MS-Search program on GC-MS computer can be used to retrieve mass spectra from NIST’02 library MS-Search program on GC-MS computer can be used to retrieve mass spectra from NIST’02 library

19 Making ions: A Practical Primer

20 Mass Spectrometer Components Inlet Inlet Get samples into the instrument Get samples into the instrument Source Ionize the molecules in a useful way Mass Analyzer Separates the ions by mass to charge (m/z) ratio Detector Detector Converts ions into an electronic signal or photons Converts ions into an electronic signal or photons Data system Data system From photographic plates to computer clusters From photographic plates to computer clusters

21 Electrospray Ionization (ESI) Dilute solution of analyte (<1 mg/L) infused through a fine needle in a high electric field Dilute solution of analyte (<1 mg/L) infused through a fine needle in a high electric field Very small, highly charged droplets are created Very small, highly charged droplets are created Solvent evaporates, droplets split and/or ions ejected to lower charge/area ratio Solvent evaporates, droplets split and/or ions ejected to lower charge/area ratio Warm nebulizing gas accelerates drying Warm nebulizing gas accelerates drying Free ions are directed into the vacuum chamber Free ions are directed into the vacuum chamber Ion source voltage depends on solvent Ion source voltage depends on solvent Usually ±2500 – ±4500 V Usually ±2500 – ±4500 V

22 Advantages of ESI Gentle ionization process Gentle ionization process High chance of observing molecular ion High chance of observing molecular ion Very labile analytes can be ionized Very labile analytes can be ionized Molecule need not be volatile Molecule need not be volatile Proteins/peptides easily analyzed by ESI Proteins/peptides easily analyzed by ESI Salts can be analyzed by ESI Salts can be analyzed by ESI Easily coupled with HPLC Easily coupled with HPLC Both positive and negative ions can be generated by the same source Both positive and negative ions can be generated by the same source

23 ESI Picture

24 Characteristics of ESI Ions ESI is a thermal process (1 atm in source) ESI is a thermal process (1 atm in source) Little fragmentation due to ionization (cf EI) Little fragmentation due to ionization (cf EI) Solution-phase ions are often preserved Solution-phase ions are often preserved e.g. organometallic salts e.g. organometallic salts ESI ions are generated by ion transfer ESI ions are generated by ion transfer (M+H) +, (M+Na) +, or (M-H) -, rarely M + or M - (M+H) +, (M+Na) +, or (M-H) -, rarely M + or M - ESI often generates multiply charged ions ESI often generates multiply charged ions (M+2H) 2+ or (M+10H) 10+ (M+2H) 2+ or (M+10H) 10+ Most ions are m/z Most ions are m/z ESI spectrum x-axis must be mass/charge (m/z or Th, not amu or Da) ESI spectrum x-axis must be mass/charge (m/z or Th, not amu or Da)

25 ESI Disadvantages Analyte must have an acidic or basic site Analyte must have an acidic or basic site Hydrocarbons and steroids not readily ionized by ESI Hydrocarbons and steroids not readily ionized by ESI Analyte must be soluble in polar, volatile solvent Analyte must be soluble in polar, volatile solvent ESI is less efficient than other sources ESI is less efficient than other sources Most ions don’t make it into the vacuum system Most ions don’t make it into the vacuum system ESI is very sensitive to contaminants ESI is very sensitive to contaminants Solvent clusters can dominate spectra Solvent clusters can dominate spectra Distribution of multiple charge states can make spectra of mixtures hard to interpret Distribution of multiple charge states can make spectra of mixtures hard to interpret e.g. polymer mass spectra e.g. polymer mass spectra

26 ESI Example I C 26 H 18 O 4 (M+H) +

27 ESI Example II 78% 22%

28 Atmospheric Pressure Chemical Ionization (APCI) APCI uses a corona discharge to generate acidic solvent cations from a vapor APCI uses a corona discharge to generate acidic solvent cations from a vapor These solvent cations can protonate hydrophobic species not amenable to ESI These solvent cations can protonate hydrophobic species not amenable to ESI APCI can be done from hexane or THF APCI can be done from hexane or THF Often used to study lipids and steroids Often used to study lipids and steroids In MSF, completely protected macrocycles are routinely studied by APCI In MSF, completely protected macrocycles are routinely studied by APCI APCI is harsher than ESI APCI is harsher than ESI Large # of variables in APCI make it less reproducible than ESI Large # of variables in APCI make it less reproducible than ESI

29 APCI Diagram

30 APCI Example

31 Agilent 6130 Multi-mode Source

32 Matrix-Assisted Laser Desorption/Ionization (MALDI) Analyte is mixed with UV-absorbing matrix Analyte is mixed with UV-absorbing matrix ~10,000:1 matrix:analyte ratio ~10,000:1 matrix:analyte ratio Analyte does not need to absorb laser Analyte does not need to absorb laser A drop of this liquid is dried on a target A drop of this liquid is dried on a target Analyte incorporated into matrix crystals Analyte incorporated into matrix crystals Spot is irradiated by a laser pulse Spot is irradiated by a laser pulse Irradiated region sublimes, taking analyte with it Irradiated region sublimes, taking analyte with it Matrix is often promoted to the excited state Matrix is often promoted to the excited state Charges exchange between matrix and analyte in the plume (very fast <100 nsec) Charges exchange between matrix and analyte in the plume (very fast <100 nsec) Ions are accelerated toward the detector Ions are accelerated toward the detector

33 MALDI Diagram Image from

34 Some Common MALDI Matrices

35 MALDI Advantages Relatively gentle ionization technique Relatively gentle ionization technique Very high MW species can be ionized Very high MW species can be ionized Molecule need not be volatile Molecule need not be volatile Very easy to get sub-picomole sensitivity Very easy to get sub-picomole sensitivity Spectra are easy to interpret Spectra are easy to interpret Positive or negative ions from same spot Positive or negative ions from same spot Wide array of matrices available Wide array of matrices available

36 MALDI Disadvantages MALDI matrix cluster ions obscure low m/z (<600) range MALDI matrix cluster ions obscure low m/z (<600) range Analyte must have very low vapor pressure Analyte must have very low vapor pressure Pulsed nature of source limits compatibility with many mass analyzers Pulsed nature of source limits compatibility with many mass analyzers Coupling MALDI with chromatography can be difficult Coupling MALDI with chromatography can be difficult Analytes that absorb the laser can be problematic Analytes that absorb the laser can be problematic Fluorescein-labeled peptides Fluorescein-labeled peptides

37 MALDI Example (Ubiq+H) + (Ins+H) + (Ubiq+2H) 2+ (ACTH 7-38+H) + (ACTH H) +

38 MALDI Example I Continued

39 Electron Ionization (EI) Gas phase molecules are irradiated by beam of energetic electrons Gas phase molecules are irradiated by beam of energetic electrons Interaction between molecule and beam results in electron ejection Interaction between molecule and beam results in electron ejection M + e -  M + + 2e - M + e -  M + + 2e - Radical species are generated initially Radical species are generated initially EI is a very energetic process EI is a very energetic process Molecules often fragment right after ionization Molecules often fragment right after ionization

40 EI Diagram Image from

41 EI Mass Spectrum Figure from Mass Spectrometry Principles and Applications E. De Hoffmann, J. Charette, V. Strooband, eds., ©1996

42 More EI Mass Spectra Cocaine Vitamin B6 Androstenedione

43 Timescales for EI-MS

44 Basic Rules Electron is first removed from site with lowest ionization potential – non-bonding electrons > pi bond electrons > sigma bond electrons – NB > π > σ (think No Pizza from Sigma) Stevenson’s Rule: During a sigma bond dissociation, the charge will likely be retained on the fragment with the lowest ionization potential Odd electron species can fragment to give odd or even electron products Even electron species can only fragment to yield even electron products Only CHARGED species are detected

45 Four Basic Mechanisms to Learn Sigma Cleavage Alpha Cleavage Inductive Cleavage McLafferty Rearrangement

46 Sigma Bond Cleavage Removal of an electron from a sigma bond weakens it As bond breaks, one fragment gets the remaining electron, and is neutral (R ) The other fragment is a charged, even electron species (R + ) Highly substituted carbocations are more stable (Stevenson’s Rule) – Cleavage of the C 1 -C 2 bond in long n-alkanes is not favored – Lower IE fragments are favored Long n-alkane chains tend to make many fragments spaced by 14 from m/z 20-90

47 Sigma Cleavage Example: Hexane 8.0 eV 8.4 eV 8.2eV

48 71 43

49 Homolytic cleavage – Radical Site Driven Cleavage is caused when an electron from a bond to an atom adjacent to the charge site pairs up with the radical – Weakened α-sigma bond breaks – This mechanism is also called α-cleavage The charge does not move in this reaction Charged product is an even electron species α-cleavage directing atoms: N > S, O, π, R > Cl, Br > H – Loss of longer alkyl chains is often favored – Energetics of both products (charged and neutral) are important

50 ΔH f = +117 kJ/mol ΔH f = +145 kJ/mol

51

52 Heterolytic Cleavage: Charge Driven Charged site induces a pair of electrons to migrate from an adjacent bond or atom – This breaks a sigma bond Also called inductive cleavage The charge migrates to the electron pair donor – The electron pair neutralizes the original charge Even electron fragments can further dissociate by this mechanism Inductive cleavage directing atoms: Halogens > O, S, >> N, C

53 136 57

54

55

56 Benzylic Bond Cleavage The charge stabilizing ability of the aromatic group can dominate EI spectra Alkylbenzenes will often form intense ions at m/z 91 – Tropylium ion – 7-membered ring favored by >11 kJ/mol Tropylium ion can fragment by successive losses of acetylene – 91  65  39 – Phenyl ions (C6H5) + decompose the same way (77  51)

57

58

59 3-Methyl-2-Pentanone

60 3-methyl-2-pentanone ions What about m/z 72?

61 McLafferty Rearrangement 72 Th fragment requires elimination of ethene A hydrogen on a carbon 4 atoms away from the carbonyl oxygen is transferred – The “1,5 shift” in carbonyl-containing ions is called the McLafferty rearrangement – Creates a distonic radical cation (charge and radical separate) – 6-membered intermediate is sterically favorable – Such rearrangements are common Once the rearrangement is complete, molecule can fragment by any previously described mechanism

62

63 EI Advantages Simplest source design of all Simplest source design of all EI mass spectrometers even go to other planets! EI mass spectrometers even go to other planets! Robust ionization mechanism Robust ionization mechanism Even noble gases are ionized by EI Even noble gases are ionized by EI Fragmentation patterns can be used to identify molecules Fragmentation patterns can be used to identify molecules NIST ’08 library has over 220,000 spectra NIST ’08 library has over 220,000 spectra Structures of novel compounds can be deduced Structures of novel compounds can be deduced

64 EI Disadvantages Fragmentation makes intact molecular ion difficult to observe Fragmentation makes intact molecular ion difficult to observe Samples must be in the gas phase Samples must be in the gas phase Databases are very limited Databases are very limited NIST’08 only has 190,000 unique compounds NIST’08 only has 190,000 unique compounds Interpreting EI spectra is an art Interpreting EI spectra is an art

65 Huygens Probe (on Titan)

66 Problem Solving with MS

67 Problem Solving Examples Formula matching with accurate mass ESI-TOF data Formula matching with accurate mass ESI-TOF data Discovery of a novel steroid (UCLA) Discovery of a novel steroid (UCLA) Diagnosing a reaction with LC-MS and accurate mass LC-MS Diagnosing a reaction with LC-MS and accurate mass LC-MS

68 Formula Matching Basics Atomic weights are not integers (except 12 C) Atomic weights are not integers (except 12 C) 14 N = Da; 11 B = Da; 1 H = Da 14 N = Da; 11 B = Da; 1 H = Da 16 O = Da; 19 F = Da; 127 I = Da 16 O = Da; 19 F = Da; 127 I = Da Difference from integer mass is called “mass defect” or “fractional mass” Difference from integer mass is called “mass defect” or “fractional mass” Related to nuclear binding energy Related to nuclear binding energy Sum of the mass defects depends on composition Sum of the mass defects depends on composition H, N increase mass defect H, N increase mass defect Hydrogen-rich molecules have high mass defects Hydrogen-rich molecules have high mass defects Eicosane (C 20 H 42 )= Eicosane (C 20 H 42 )= O, Cl, F, Na decrease it O, Cl, F, Na decrease it Hydrogen deficient species have low mass defects Hydrogen deficient species have low mass defects Morphine, (C 17 H 19 NO 3 ) = Morphine, (C 17 H 19 NO 3 ) =

69 More Formula Matching Accurate mass measurements narrow down possible formulas for a given molecular weight Accurate mass measurements narrow down possible formulas for a given molecular weight 534 entries in NIST’08 mass entries in NIST’08 mass 285 Only 3 formulas within 5 ppm of Only 3 formulas within 5 ppm of compounds with formula C 17 H 19 NO 3 46 compounds with formula C 17 H 19 NO 3 Mass spectrum and user info complete the picture Mass spectrum and user info complete the picture Isotope distributions indicate/eliminate elements Isotope distributions indicate/eliminate elements User-supplied info eliminates others (e.g. no F) User-supplied info eliminates others (e.g. no F) Suggested formula has to make chemical sense Suggested formula has to make chemical sense

70 Formula Matching Example Only 9 ways to combine up to 40 C, 50 H, 5 N, 5 O, and 2 Cl to get a mass within 20 ppm ( u) of , only 3 have 2 Cl Zoloft C 17 H 18 Cl 2 N

71 Discovery of a Novel Steroid A researcher at UCLA was given an athlete’s used syringe that contained a suspected steroid A researcher at UCLA was given an athlete’s used syringe that contained a suspected steroid GC-MS revealed a mass spectrum that matched no known steroid GC-MS revealed a mass spectrum that matched no known steroid Compound was NOT detected by normal steroid screen Compound was NOT detected by normal steroid screen The mass spectrum was similar to two other steroids The mass spectrum was similar to two other steroids Accurate mass spectrometry indicated a molecular formula of C 21 H 28 O 2 ( Da) Accurate mass spectrometry indicated a molecular formula of C 21 H 28 O 2 ( Da) Rapid Communications in Mass Spectrometry vol. 18, page 1245 (2004)

72 Unknown mass spectrum Trenbalone mass spectrum Trenbalone is used to aid growth in US beef cattle Gestrinone mass spectrum Used in Europe to treat endometriosis New molecule dubbed THG or tetrahydrogestrinone is active ingredient in “The Clear”

73 Staudinger Rxn Gone Wrong? M+H for product is M+Na is

74 LC-MS Chromatogram

75 Mass Spectra from Peaks

76 Mass Spectra from Peaks 2

77 Proposed Side Reaction

78 Accurate Mass Data M+H for deuterated amine is (-40 ppm)


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