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Mass spectrometry

Mass spectrometry is a technique that uses high energy electrons to break molecules into fragments. It then measures the masses of the fragments to reveal information about the molecular structure. Key aspects of mass spectrometry include the ionization source, mass analyzer, and detector. Common ionization methods are electron impact, electrospray, and MALDI, with softer methods like electrospray and MALDI used for larger molecules like proteins. Mass analyzers separate the ions by mass to charge ratio and include quadrupoles, time-of-flight, and magnetic sectors. The detector then counts the ions to produce a mass spectrum.

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By Kommineni.vidyachowdhary M.pharm Vaagdevi pharmacy college warangal 1 MASS SPECTROMETRYMASS SPECTROMETRY
Mass spectrometry is the most accurate method for determining the molecular mass of the compound and its elemental composition. It is also called as positive ion spectra or line spectra. 06/13/15 2
Introduction • Mass spectrometry (Mass Spec or MS) uses high energy electrons to break a molecule into fragments. • It does not involve the absorption or emission of light. • A beam of high-energy electrons breaks the molecule apart. • The masses of the fragments and their relative abundance reveal information about the structure of the molecule. • Separation and analysis of the fragments provides information about: – Molecular weight – Structure
Mass Spec Principles IonizerIonizer Sample + _ Mass AnalyzerMass Analyzer DetectorDetector
06/13/15 5 Schematic of Mass Spectrometry Ionizer ↓ Mass-to-charge ratio Analyzer ↓ Detector
How does a mass spectrometer work? • Ionization method – MALDI – Electrospray (Proteins must be charged and dry) • Mass analyzer – MALDI-TOF • MW – Triple Quadrapole • AA seq – MALDI-QqTOF • AA seq and MW – QqTOF • AA seq and protein modif. Create ions Separate ions Detect ions • Mass spectrum • Database analysis
06/13/15 7 What is a Mass Spectrometer? A Mass Spectrometer is a machine that weighs molecules ! (by measuring the mass to charge ratio of ions) Source EI CI ESI APCI APPI MALDI Dispersion TOF FT-ICR Sector Quad Trap Detector Faraday Cup Channeltron MCP
06/13/15 8 Mass Spectrometry Categorization • Based on ionization: Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry Electrospray ionization (ESI) mass spectrometry Induction-coupled plasma mass spectrometry (ICP-MS) Atmospheric Ionization Mass Spectrometry Secondary Ionization Mass Spectrometry (SIMS) • Based on M/Z Separation: Quadrupole Mass spectrometry Ion trap mass spectrometry magnetic sector mass spectrometry time-of-flight mass spectrometry Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Ion Mobility Mass Spectrometry
06/13/15 9 Mass Spectrometry Categorization (continued) • Based on Applications: Environmental Mass Spectrometry Biological Mass Spectrometry Cell Mass Spectrometry Portable Mass Spectrometry • Based on Configuration: Tandem Mass Spectrometry • Based on Sample Introduction: GCMS; LCMS, Electrophoresis Mass Spectrometry
06/13/15 10 MS Operation • Nearly all mass spectrometers need to operate under high vacuum condition with the pressure less than 10 -5 Torr with the only exceptions of an ion trap mass spectrometer (milli-Torr) and an ion mobility mass spectrometer (Torr). • Never turn on a mass spectrometer without knowing the chamber pressure. • A tour to major mass spectrometry facilities in Genomic Research Center, Sinica will be arranged.
Mass spectrometersMass spectrometersL in e a r T im e O f F lig h t tu b e R e fle c t o r T im e O f F lig h t t u b e d e te c to r re fle c to r io n s o u rc e io n s o u rc e d e te c to r tim e o f flig h t tim e o f flig h t • Time of flight (TOF) (MALDI)Time of flight (TOF) (MALDI) – Measures the time required for ions to fly down the length of a chamber. – Often combined with MALDI (MALDI-TOF) Detections from multiple laser bursts are averaged. Multiple laser • Tandem MS- MS/MSTandem MS- MS/MS -separation and identification of compounds in complex mixtures - induce fragmentation and mass analyze the fragment ions. - Uses two or more mass analyzers/filters separated by a collision cell filled with Argon or Xenon • Different MS-MS configurations – Quadrupole-quadrupole (low energy) – Magnetic sector-quadrupole (high) – Quadrupole-time-of-flight (low energy) – Time-of-flight-time-of-flight (low energy)
Typical Mass Spectrometer
LC/LC-MS/MS-Tandem LC, Tandem MS
Typical Mass Spectrum • Characterized by sharp, narrow peaks • X-axis position indicates the m/z ratio of a given ion (for singly charged ions this corresponds to the mass of the ion) • Height of peak indicates the relative abundance of a given ion (not reliable for quantitation) • Peak intensity indicates the ion’s ability to desorb or “fly” (some fly better than others)
m/z ratio: Molecular weight divided by the Charge on this protein All proteins are sorted based on a mass to charge ratio (m/z)
Typical Mass Spectrum aspirin RelativeRelative AbundanceAbundance 120 m/z-for singlysingly charged ion this is the mass
Resolution & Resolving PowerResolution & Resolving Power • Width of peak indicates the resolution of the MS instrument • The better the resolution or resolving power, the better the instrument and the better the mass accuracy • Resolving power is defined as: M is the mass number of the observed mass (∆M) is the difference between two masses that can be separated
Resolution in MSResolution in MS
Resolution in MSResolution in MS QTOF 783.455 784.465 785.475 783.6
06/13/15 20
Introduction to Mass Spectrometry Sample introduction Ionization Minimize collisions, interferences Separate masses Count ions Collect results Nier-type mass spec
The sample cone isolates the torch from the interior. The torch box of an Agilent 7500 ICPMS spray chamber torchAr feed RF coil
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N SSource magnets Filament Collector Sample Inlet + + + + + + ++ + + + + + + + + Extraction lenses Electron ionization 06/13/15 25
In this type anode and cathode are arranged with a very fine gap (0.5 to 2mm) which may serve as a slit. The gaseous sample introduced at the anode points where the electric field is concentrated. The ionisation of the sample takes place by extraction of electrons from the sample by microtips of the anode. 06/13/15 26
N SSource magnets Filament Collector Sample Inlet + + + + + + ++ + + + + + + + + Extraction lenses + ++ + + + + + + + + + + + Chemical ionization 06/13/15 27
These are the devices used to separate the ions produced in the ion source into their individual m/z ratios and focus them on the detector. A number of different mass analysers exist in mass spectrometry. 06/13/15 28
 Magnetic sector – single focussing - double focussing  Quadrupole analysers  ion trap (Quistor) devices  tiMe-of-flight (tof) analysers 06/13/15 29
Magnetic sectors – single focussing •Generally bulky and expensive. •Earliest type of analyser and still popular. •High accelerating potential especially in comparison with other methods (usually 4- 10kV). •Magnets wedge shaped and must provide homogeneous fields. •Ion beam enters and exits at exactly 90˚. •Focuses ions according to their momentum. 06/13/15 30
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Magnetic sectors – double focussing •Adds a second electric sector to provide energy focussing of ions independent of mass. •Double focussing means that both the energy and momentum focus is designed to coincide at the collector slit. •Very high mass resolution can be achieved by this arrangement. •Very bulky and expensive but high performance. 06/13/15 32
06/13/15 33
Quadrupole Mass Analyzer 06/13/15 34 + + + +
The quadrupole consists of four parallel rods. The opposing rods have the same polarity whilst adjacent rods have opposite polarity. Each rod is applied with a DC and an RF voltage.Ions are scanned by varying the DC/Rf quadrupole voltages. Only ions with the selected mass to charge ratio will have the correct oscillatory pathway in the Rf field. 06/13/15 35
• Consists of ring electrode and two end caps • Principle very similar to quadrupole • Ions stored by RF & DC fields • Scanning field can eject ions of specific m/z • Advantages - MS/MS/MS….. - High sensitivity full scan MS/MS 06/13/15 36
06/13/15 37
• In this type of analyser the sorting of the ions is done in absence of magnetic field. • It operates on the principle that, if the ions produced are supplied with equal energy and allowed to travel predetermined distance then they will acquire different velocities depending of their masses. 06/13/15 38
• The detector records the charge induced when an ion passes by or hits a surface • Electron Multipliers (EM)* – Most common detector – -Can Detect positive and negative ions 06/13/15 39
• Faraday Cup – Least expensive detector – Captured ions transfer charge to cup – used to calibrate other MS detectors 06/13/15 40
• Photographic detection: This detector system is most sensitive than any other detector because the photoplate integrates the ion signal over a period of time. The photoplates are processed by the usual photographic techniques and read with the aid of densitometer. 06/13/15 41
Different Ionization Methods • Electron Impact (EI - Hard method) – small molecules, 1-1000 Daltons, structure • Fast Atom Bombardment (FAB – Semi-hard) – peptides, sugars, up to 6000 Daltons • Electrospray Ionization (ESI - Soft) – peptides, proteins, up to 200,000 Daltons • Matrix Assisted Laser Desorption (MALDI-Soft) – peptides, proteins, DNA, up to 500 kD
Electron Impact IonizationElectron Impact Ionization • Sample introduced into instrument by heating it until it evaporates • Gas phase sample is bombarded with electrons coming from rhenium or tungsten filament (energy = 70 eV) • Molecule is “shattered” into fragments (70 eV >> 5 eV bonds) • Fragments sent to mass analyzer
EI Fragmentation of CHEI Fragmentation of CH33OHOH CH3OH CH3OH+ CH3OH CH2O=H+ + H CH3OH + CH3 + OH CHO=H+ + HCH2O=H+ Why wouldn’t Electron Impact be suitable for analyzing proteins?
Why You Can’t Use EI For AnalyzingWhy You Can’t Use EI For Analyzing ProteinsProteins • EI shatters chemical bonds • Any given protein contains 20 different amino acids • EI would shatter the protein into not only into amino acids but also amino acid sub-fragments and even peptides of 2,3,4… amino acids • Result is 10,000’s of different signals from a single protein -- too complex to analyze
Soft Ionization MethodsSoft Ionization Methods 337 nm UV laser MALDI cyano-hydroxy cinnamic acid Gold tip needle Fluid (no salt) ESI + _
Soft IonizationSoft Ionization • Soft ionization techniques keep the molecule of interest fully intact • Electro-spray ionization first conceived in 1960’s by Malcolm Dole but put into practice in 1980’s by John Fenn (Yale) • MALDI first introduced in 1985 by Franz Hillenkamp and Michael Karas (Frankfurt) • Made it possible to analyze large molecules via inexpensive mass analyzers such as quadrupole, ion trap and TOF
Ionization methodsIonization methods • Electrospray mass spectrometry (ESI-MS) – Liquid containing analyte is forced through a steel capillary at high voltage to electrostatically disperse analyte. Charge imparted from rapidly evaporating liquid. • Matrix-assisted laser desorption ionization (MALDI) – Analyte (protein) is mixed with large excess of matrix (small organic molecule) – Irradiated with short pulse of laser light. Wavelength of laser is the same as absorbance max of matrix.
Electrospray IonizationElectrospray Ionization • Sample dissolved in polar, volatile buffer (no salts) and pumped through a stainless steel capillary (70 - 150 µm) at a rate of 10-100 µL/min • Strong voltage (3-4 kV) applied at tip along with flow of nebulizing gas causes the sample to “nebulize” or aerosolize • Aerosol is directed through regions of higher vacuum until droplets evaporate to near atomic size (still carrying charges)
Electrospray (Detail)Electrospray (Detail)
Electrospray IonizationElectrospray Ionization • Can be modified to “nanospray” system with flow < 1 µL/min • Very sensitive technique, requires less than a picomole of material • Strongly affected by salts & detergents • Positive ion mode measures (M + H)+ (add formic acid to solvent) • Negative ion mode measures (M - H)- (add ammonia to solvent)
Positive or Negative Ion Mode?Positive or Negative Ion Mode? • If the sample has functional groups that readily accept H+ (such as amide and amino groups found in peptides and proteins) then positive ion detection is used-PROTEINS • If a sample has functional groups that readily lose a proton (such as carboxylic acids and hydroxyls as found in nucleic acids and sugars) then negative ion detection is used-DNA
Matrix-Assisted Laser DesorptionMatrix-Assisted Laser Desorption IonizationIonization 337 nm UV laser MALDI cyano-hydroxy cinnamic acid
MALDIMALDI • Sample is ionized by bombarding sample with laser light • Sample is mixed with a UV absorbant matrix (sinapinic acid for proteins, 4-hydroxycinnaminic acid for peptides) • Light wavelength matches that of absorbance maximum of matrix so that the matrix transfers some of its energy to the analyte (leads to ion sputtering)
HT Spotting on a MALDI Plate
MALDI IonizationMALDI Ionization + + + + - - - + + + + - - --+ + Analyte Matrix Laser + + + • Absorption of UV radiation by chromophoric matrix and ionization of matrix • Dissociation of matrix, phase change to super-compressed gas, charge transfer to analyte molecule • Expansion of matrix at supersonic velocity, analyte trapped in expanding matrix plume (explosion/”popping”) + + +
MALDIMALDI • Unlike ESI, MALDI generates spectra that have just a singly charged ion • Positive mode generates ions of M + H • Negative mode generates ions of M - H • Generally more robust that ESI (tolerates salts and nonvolatile components) • Easier to use and maintain, capable of higher throughput • Requires 10 µL of 1 pmol/µL sample
Principal for MALDI-TOF MASSPrincipal for MALDI-TOF MASS + + ++ ++ + + + + p u ls e d U V o r IR la s e r ( 3 - 4 n s ) d e te c to r v a c u u m s t r o n g e le c tr ic fie ld T im e O f F lig h t t u b e p e p tid e m ix tu r e e m b e d d e d in lig h t a b s o r b in g c h e m ic a ls ( m a tr ix ) c lo u d o f p r o to n a te d p e p tid e m o le c u le sa c cV
Principal for MALDI-TOF MASSPrincipal for MALDI-TOF MASS L in e a r T i m e O f F li g h t t u b e R e f le c t o r T im e O f F l ig h t t u b e d e t e c t o r r e f le c t o r io n s o u r c e io n s o u r c e d e t e c t o r t im e o f flig h t t im e o f flig h t
MALDIMALDI == SELDISELDI 337 nm UV laser MALDI cyano-hydroxy cinnaminic acid
MALDI/SELDI SpectraMALDI/SELDI Spectra Normal Tumor
Background of fragmentation • The impact of a stream of high energy electrons causes the molecule to lose an electron forming a radical cation. – A species with a positive charge and one unpaired electron + e - C H H H H H H H HC + 2 e - Molecular ion (M+ ) m/z = 16
Background • The impact of the stream of high energy electrons can also break the molecule or the radical cation into fragments. (not detected by MS) m/z = 29 molecular ion (M + ) m/z = 30 + C H H H + H HH C H H C H H H C H H C H H H C H H + e - H C H H C H H H m/z = 15
Background • Molecular ion (parent ion): – The radical cation corresponding to the mass of the original molecule • The molecular ion is usually the highest mass in the spectrum – Some exceptions w/specific isotopes – Some molecular ion peaks are absent. H H H HC H C H H C H H H
Background • Mass spectrum of ethanol (MW = 46) M+
Background • The cations that are formed are separated by magnetic deflection.
Background • Only cations are detected. – Radicals are “invisible” in MS. • The amount of deflection observed depends on the mass to charge ratio (m/z). – Most cations formed have a charge of +1 so the amount of deflection observed is usually dependent on the mass of the ion.
Background • The resulting mass spectrum is a graph of the mass of each cation vs. its relative abundance. • The peaks are assigned an abundance as a percentage of the base peak. – the most intense peak in the spectrum • The base peak is not necessarily the same as the parent ion peak.
Background M+ base peak The mass spectrum of ethanol
Background • Most elements occur naturally as a mixture of isotopes. – The presence of significant amounts of heavier isotopes leads to small peaks that have masses that are higher than the parent ion peak. • M+1 = a peak that is one mass unit higher than M+ • M+2 = a peak that is two mass units higher than M+
Easily Recognized Elements in MS • Nitrogen: – Odd number of N = odd MW CH3CN M + = 41 SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/2/09)
Easily Recognized Elements in MS  Bromine:  M+ ~ M+2 (50.5% 79 Br/49.5% 81 Br) 2-bromopropane M+ ~ M+2 SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/1/09)
Easily Recognized Elements in MS • Chlorine: – M+2 is ~ 1/3 as large as M+ Cl SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/2/09) M+2 M+
• Sulfur: – M+2 larger than usual (4% of M+ ) Easily Recognized Elements in MS M+ Unusually large M+2 S SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/1/09)
Easily Recognized Elements in MS • Iodine – I+ at 127 – Large gap Large gap I+ M+ SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/2/09) ICH2CN
Fragmentation Patterns • The impact of the stream of high energy electrons often breaks the molecule into fragments, commonly a cation and a radical. – Bonds break to give the most stable cation. – Stability of the radical is less important.
Fragmentation Patterns • Alkanes – Fragmentation often splits off simple alkyl groups: • Loss of methyl M+ - 15 • Loss of ethyl M+ - 29 • Loss of propyl M+ - 43 • Loss of butyl M+ - 57 – Branched alkanes tend to fragment forming the most stable carbocations.
Fragmentation Patterns • Mass spectrum of 2-methylpentane
Fragmentation Patterns • Alkenes: – Fragmentation typically forms resonance stabilized allylic carbocations
Fragmentation Patterns • Aromatics: – Fragment at the benzylic carbon, forming a resonance stabilized benzylic carbocation (which rearranges to the tropylium ion) M+ CH H CH Br H C H H or
Fragmentation Patterns • Alcohols – Fragment easily resulting in very small or missing parent ion peak – May lose hydroxyl radical or water • M+ - 17 or M+ - 18 – Commonly lose an alkyl group attached to the carbinol carbon forming an oxonium ion. • 1o alcohol usually has prominent peak at m/z = 31 corresponding to H2C=OH+
Fragmentation Patterns • MS for 1-propanol M+ M+ -18 CH3CH2CH2OH H2C OH SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/28/09)
Fragmentation Patterns • Amines – Odd M+ (assuming an odd number of nitrogens are present) – α-cleavage dominates forming an iminium ion CH3CH2 CH2 N H CH2 CH2CH2CH3 CH3CH2CH2N CH2 H m/z =72 iminium ion
Fragmentation Patterns 86 CH3CH2 CH2 N H CH2 CH2CH2CH3 72
Fragmentation Patterns • Ethers – α-cleavage forming oxonium ion – Loss of alkyl group forming oxonium ion – Loss of alkyl group forming a carbocation
Fragmentation Patterns H O CHCH3 MS of diethylether (CH3CH2OCH2CH3) CH3CH2O CH2 H O CH2
Fragmentation Patterns • The impact of the stream of high energy electrons often breaks the molecule into fragments, commonly a cation and a radical. – Bonds break to give the most stable cation. – Stability of the radical is less important.
Fragmentation Patterns • Alkanes – Fragmentation often splits off simple alkyl groups: • Loss of methyl M+ - 15 • Loss of ethyl M+ - 29 • Loss of propyl M+ - 43 • Loss of butyl M+ - 57 – Branched alkanes tend to fragment forming the most stable carbocations.
Fragmentation Patterns • Mass spectrum of 2-methylpentane
Fragmentation Patterns • Alkenes: – Fragmentation typically forms resonance stabilized allylic carbocations
Fragmentation Patterns • Aromatics: – Fragment at the benzylic carbon, forming a resonance stabilized benzylic carbocation (which rearranges to the tropylium ion) M+ CH H CH Br H C H H or
Fragmentation Patterns Aromatics may also have a peak at m/z = 77 for the benzene ring. NO2 77 M+ = 123 77
Fragmentation Patterns • Alcohols – Fragment easily resulting in very small or missing parent ion peak – May lose hydroxyl radical or water • M+ - 17 or M+ - 18 – Commonly lose an alkyl group attached to the carbinol carbon forming an oxonium ion. • 1o alcohol usually has prominent peak at m/z = 31 corresponding to H2C=OH+
Fragmentation Patterns • MS for 1-propanol M+ M+ -18 CH3CH2CH2OH H2C OH SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/28/09)
Fragmentation Patterns • Amines – Odd M+ (assuming an odd number of nitrogens are present) – α-cleavage dominates forming an iminium ion CH3CH2 CH2 N H CH2 CH2CH2CH3 CH3CH2CH2N CH2 H m/z =72 iminium ion
Fragmentation Patterns 86 CH3CH2 CH2 N H CH2 CH2CH2CH3 72
Fragmentation Patterns • Ethers – α-cleavage forming oxonium ion – Loss of alkyl group forming oxonium ion – Loss of alkyl group forming a carbocation
Fragmentation Patterns • Aldehydes (RCHO) – Fragmentation may form acylium ion – Common fragments: • M+ - 1 for • M+ - 29 for RC O R (i.e. RCHO - CHO) RC O
Fragmentation Patterns • MS for hydrocinnamaldehyde M+ = 134 C C C H H H H H O 133 105 91 105 91 SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/28/09)
Fragmentation Patterns • Ketones – Fragmentation leads to formation of acylium ion: • Loss of R forming • Loss of R’ forming RC O R'C O RCR' O
Fragmentation Patterns • MS for 2-pentanone CH3CCH2CH2CH3 O M+ CH3CH2CH2C O CH3C O SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/28/09)
Fragmentation Patterns • Esters (RCO2R’) – Common fragmentation patterns include: • Loss of OR’ – peak at M+ - OR’ • Loss of R’ – peak at M+ - R’
Frgamentation Patterns M+ = 136 C O O CH3 105 77 105 77 SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/28/09)
Rule of Thirteen • The “Rule of Thirteen” can be used to identify possible molecular formulas for an unknown hydrocarbon, CnHm. – Step 1: n = M+ /13 (integer only, use remainder in step 2) – Step 2: m = n + remainder from step 1
Rule of Thirteen • Example: The formula for a hydrocarbon with M+ =106 can be found: – Step 1: n = 106/13 = 8 (R = 2) – Step 2: m = 8 + 2 = 10 – Formula: C8H10
Rule of Thirteen • If a heteroatom is present, – Subtract the mass of each heteroatom from the MW – Calculate the formula for the corresponding hydrocarbon – Add the heteroatoms to the formula
Rule of Thirteen Example: A compound with a molecular ion peak at m/z = 102 has a strong peak at 1739 cm-1 in its IR spectrum. Determine its molecular formula.
References • Chatwal GR, Anand SK. Instrumental method of chemical analysis, Himalaya publishing house. • Sharma YR. Elementary organic spectroscopy. • Willard,merritt,dean. Instumental methods of analysis. • www.google.com/images 06/13/15 110
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Mass spectrometry

  • 1.
    By Kommineni.vidyachowdhary M.pharm Vaagdevi pharmacycollege warangal 1 MASS SPECTROMETRYMASS SPECTROMETRY
  • 2.
    Mass spectrometry isthe most accurate method for determining the molecular mass of the compound and its elemental composition. It is also called as positive ion spectra or line spectra. 06/13/15 2
  • 3.
    Introduction • Mass spectrometry(Mass Spec or MS) uses high energy electrons to break a molecule into fragments. • It does not involve the absorption or emission of light. • A beam of high-energy electrons breaks the molecule apart. • The masses of the fragments and their relative abundance reveal information about the structure of the molecule. • Separation and analysis of the fragments provides information about: – Molecular weight – Structure
  • 4.
    Mass Spec Principles IonizerIonizer Sample + _ MassAnalyzerMass Analyzer DetectorDetector
  • 5.
    06/13/15 5 Schematic ofMass Spectrometry Ionizer ↓ Mass-to-charge ratio Analyzer ↓ Detector
  • 6.
    How does amass spectrometer work? • Ionization method – MALDI – Electrospray (Proteins must be charged and dry) • Mass analyzer – MALDI-TOF • MW – Triple Quadrapole • AA seq – MALDI-QqTOF • AA seq and MW – QqTOF • AA seq and protein modif. Create ions Separate ions Detect ions • Mass spectrum • Database analysis
  • 7.
    06/13/15 7 What isa Mass Spectrometer? A Mass Spectrometer is a machine that weighs molecules ! (by measuring the mass to charge ratio of ions) Source EI CI ESI APCI APPI MALDI Dispersion TOF FT-ICR Sector Quad Trap Detector Faraday Cup Channeltron MCP
  • 8.
    06/13/15 8 Mass SpectrometryCategorization • Based on ionization: Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry Electrospray ionization (ESI) mass spectrometry Induction-coupled plasma mass spectrometry (ICP-MS) Atmospheric Ionization Mass Spectrometry Secondary Ionization Mass Spectrometry (SIMS) • Based on M/Z Separation: Quadrupole Mass spectrometry Ion trap mass spectrometry magnetic sector mass spectrometry time-of-flight mass spectrometry Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Ion Mobility Mass Spectrometry
  • 9.
    06/13/15 9 Mass SpectrometryCategorization (continued) • Based on Applications: Environmental Mass Spectrometry Biological Mass Spectrometry Cell Mass Spectrometry Portable Mass Spectrometry • Based on Configuration: Tandem Mass Spectrometry • Based on Sample Introduction: GCMS; LCMS, Electrophoresis Mass Spectrometry
  • 10.
    06/13/15 10 MS Operation •Nearly all mass spectrometers need to operate under high vacuum condition with the pressure less than 10 -5 Torr with the only exceptions of an ion trap mass spectrometer (milli-Torr) and an ion mobility mass spectrometer (Torr). • Never turn on a mass spectrometer without knowing the chamber pressure. • A tour to major mass spectrometry facilities in Genomic Research Center, Sinica will be arranged.
  • 11.
    Mass spectrometersMass spectrometersLin e a r T im e O f F lig h t tu b e R e fle c t o r T im e O f F lig h t t u b e d e te c to r re fle c to r io n s o u rc e io n s o u rc e d e te c to r tim e o f flig h t tim e o f flig h t • Time of flight (TOF) (MALDI)Time of flight (TOF) (MALDI) – Measures the time required for ions to fly down the length of a chamber. – Often combined with MALDI (MALDI-TOF) Detections from multiple laser bursts are averaged. Multiple laser • Tandem MS- MS/MSTandem MS- MS/MS -separation and identification of compounds in complex mixtures - induce fragmentation and mass analyze the fragment ions. - Uses two or more mass analyzers/filters separated by a collision cell filled with Argon or Xenon • Different MS-MS configurations – Quadrupole-quadrupole (low energy) – Magnetic sector-quadrupole (high) – Quadrupole-time-of-flight (low energy) – Time-of-flight-time-of-flight (low energy)
  • 12.
  • 13.
  • 14.
    Typical Mass Spectrum •Characterized by sharp, narrow peaks • X-axis position indicates the m/z ratio of a given ion (for singly charged ions this corresponds to the mass of the ion) • Height of peak indicates the relative abundance of a given ion (not reliable for quantitation) • Peak intensity indicates the ion’s ability to desorb or “fly” (some fly better than others)
  • 15.
    m/z ratio: Molecular weightdivided by the Charge on this protein All proteins are sorted based on a mass to charge ratio (m/z)
  • 16.
    Typical Mass Spectrum aspirin RelativeRelative AbundanceAbundance 120m/z-for singlysingly charged ion this is the mass
  • 17.
    Resolution & ResolvingPowerResolution & Resolving Power • Width of peak indicates the resolution of the MS instrument • The better the resolution or resolving power, the better the instrument and the better the mass accuracy • Resolving power is defined as: M is the mass number of the observed mass (∆M) is the difference between two masses that can be separated
  • 18.
  • 19.
    Resolution in MSResolutionin MS QTOF 783.455 784.465 785.475 783.6
  • 20.
  • 21.
    Introduction to MassSpectrometry Sample introduction Ionization Minimize collisions, interferences Separate masses Count ions Collect results Nier-type mass spec
  • 22.
    The sample coneisolates the torch from the interior. The torch box of an Agilent 7500 ICPMS spray chamber torchAr feed RF coil
  • 23.
  • 24.
  • 25.
  • 26.
    In this typeanode and cathode are arranged with a very fine gap (0.5 to 2mm) which may serve as a slit. The gaseous sample introduced at the anode points where the electric field is concentrated. The ionisation of the sample takes place by extraction of electrons from the sample by microtips of the anode. 06/13/15 26
  • 27.
  • 28.
    These are thedevices used to separate the ions produced in the ion source into their individual m/z ratios and focus them on the detector. A number of different mass analysers exist in mass spectrometry. 06/13/15 28
  • 29.
     Magnetic sector– single focussing - double focussing  Quadrupole analysers  ion trap (Quistor) devices  tiMe-of-flight (tof) analysers 06/13/15 29
  • 30.
    Magnetic sectors –single focussing •Generally bulky and expensive. •Earliest type of analyser and still popular. •High accelerating potential especially in comparison with other methods (usually 4- 10kV). •Magnets wedge shaped and must provide homogeneous fields. •Ion beam enters and exits at exactly 90˚. •Focuses ions according to their momentum. 06/13/15 30
  • 31.
  • 32.
    Magnetic sectors –double focussing •Adds a second electric sector to provide energy focussing of ions independent of mass. •Double focussing means that both the energy and momentum focus is designed to coincide at the collector slit. •Very high mass resolution can be achieved by this arrangement. •Very bulky and expensive but high performance. 06/13/15 32
  • 33.
  • 34.
  • 35.
    The quadrupole consistsof four parallel rods. The opposing rods have the same polarity whilst adjacent rods have opposite polarity. Each rod is applied with a DC and an RF voltage.Ions are scanned by varying the DC/Rf quadrupole voltages. Only ions with the selected mass to charge ratio will have the correct oscillatory pathway in the Rf field. 06/13/15 35
  • 36.
    • Consists ofring electrode and two end caps • Principle very similar to quadrupole • Ions stored by RF & DC fields • Scanning field can eject ions of specific m/z • Advantages - MS/MS/MS….. - High sensitivity full scan MS/MS 06/13/15 36
  • 37.
  • 38.
    • In thistype of analyser the sorting of the ions is done in absence of magnetic field. • It operates on the principle that, if the ions produced are supplied with equal energy and allowed to travel predetermined distance then they will acquire different velocities depending of their masses. 06/13/15 38
  • 39.
    • The detectorrecords the charge induced when an ion passes by or hits a surface • Electron Multipliers (EM)* – Most common detector – -Can Detect positive and negative ions 06/13/15 39
  • 40.
    • Faraday Cup –Least expensive detector – Captured ions transfer charge to cup – used to calibrate other MS detectors 06/13/15 40
  • 41.
    • Photographic detection: Thisdetector system is most sensitive than any other detector because the photoplate integrates the ion signal over a period of time. The photoplates are processed by the usual photographic techniques and read with the aid of densitometer. 06/13/15 41
  • 42.
    Different Ionization Methods •Electron Impact (EI - Hard method) – small molecules, 1-1000 Daltons, structure • Fast Atom Bombardment (FAB – Semi-hard) – peptides, sugars, up to 6000 Daltons • Electrospray Ionization (ESI - Soft) – peptides, proteins, up to 200,000 Daltons • Matrix Assisted Laser Desorption (MALDI-Soft) – peptides, proteins, DNA, up to 500 kD
  • 43.
    Electron Impact IonizationElectronImpact Ionization • Sample introduced into instrument by heating it until it evaporates • Gas phase sample is bombarded with electrons coming from rhenium or tungsten filament (energy = 70 eV) • Molecule is “shattered” into fragments (70 eV >> 5 eV bonds) • Fragments sent to mass analyzer
  • 45.
    EI Fragmentation ofCHEI Fragmentation of CH33OHOH CH3OH CH3OH+ CH3OH CH2O=H+ + H CH3OH + CH3 + OH CHO=H+ + HCH2O=H+ Why wouldn’t Electron Impact be suitable for analyzing proteins?
  • 46.
    Why You Can’tUse EI For AnalyzingWhy You Can’t Use EI For Analyzing ProteinsProteins • EI shatters chemical bonds • Any given protein contains 20 different amino acids • EI would shatter the protein into not only into amino acids but also amino acid sub-fragments and even peptides of 2,3,4… amino acids • Result is 10,000’s of different signals from a single protein -- too complex to analyze
  • 47.
    Soft Ionization MethodsSoftIonization Methods 337 nm UV laser MALDI cyano-hydroxy cinnamic acid Gold tip needle Fluid (no salt) ESI + _
  • 48.
    Soft IonizationSoft Ionization •Soft ionization techniques keep the molecule of interest fully intact • Electro-spray ionization first conceived in 1960’s by Malcolm Dole but put into practice in 1980’s by John Fenn (Yale) • MALDI first introduced in 1985 by Franz Hillenkamp and Michael Karas (Frankfurt) • Made it possible to analyze large molecules via inexpensive mass analyzers such as quadrupole, ion trap and TOF
  • 50.
    Ionization methodsIonization methods •Electrospray mass spectrometry (ESI-MS) – Liquid containing analyte is forced through a steel capillary at high voltage to electrostatically disperse analyte. Charge imparted from rapidly evaporating liquid. • Matrix-assisted laser desorption ionization (MALDI) – Analyte (protein) is mixed with large excess of matrix (small organic molecule) – Irradiated with short pulse of laser light. Wavelength of laser is the same as absorbance max of matrix.
  • 51.
    Electrospray IonizationElectrospray Ionization •Sample dissolved in polar, volatile buffer (no salts) and pumped through a stainless steel capillary (70 - 150 µm) at a rate of 10-100 µL/min • Strong voltage (3-4 kV) applied at tip along with flow of nebulizing gas causes the sample to “nebulize” or aerosolize • Aerosol is directed through regions of higher vacuum until droplets evaporate to near atomic size (still carrying charges)
  • 52.
  • 53.
    Electrospray IonizationElectrospray Ionization •Can be modified to “nanospray” system with flow < 1 µL/min • Very sensitive technique, requires less than a picomole of material • Strongly affected by salts & detergents • Positive ion mode measures (M + H)+ (add formic acid to solvent) • Negative ion mode measures (M - H)- (add ammonia to solvent)
  • 54.
    Positive or NegativeIon Mode?Positive or Negative Ion Mode? • If the sample has functional groups that readily accept H+ (such as amide and amino groups found in peptides and proteins) then positive ion detection is used-PROTEINS • If a sample has functional groups that readily lose a proton (such as carboxylic acids and hydroxyls as found in nucleic acids and sugars) then negative ion detection is used-DNA
  • 55.
    Matrix-Assisted Laser DesorptionMatrix-AssistedLaser Desorption IonizationIonization 337 nm UV laser MALDI cyano-hydroxy cinnamic acid
  • 56.
    MALDIMALDI • Sample isionized by bombarding sample with laser light • Sample is mixed with a UV absorbant matrix (sinapinic acid for proteins, 4-hydroxycinnaminic acid for peptides) • Light wavelength matches that of absorbance maximum of matrix so that the matrix transfers some of its energy to the analyte (leads to ion sputtering)
  • 57.
    HT Spotting ona MALDI Plate
  • 58.
    MALDI IonizationMALDI Ionization + + + + - - - + + + + - - --+ + Analyte Matrix Laser + + + •Absorption of UV radiation by chromophoric matrix and ionization of matrix • Dissociation of matrix, phase change to super-compressed gas, charge transfer to analyte molecule • Expansion of matrix at supersonic velocity, analyte trapped in expanding matrix plume (explosion/”popping”) + + +
  • 59.
    MALDIMALDI • Unlike ESI,MALDI generates spectra that have just a singly charged ion • Positive mode generates ions of M + H • Negative mode generates ions of M - H • Generally more robust that ESI (tolerates salts and nonvolatile components) • Easier to use and maintain, capable of higher throughput • Requires 10 µL of 1 pmol/µL sample
  • 60.
    Principal for MALDI-TOFMASSPrincipal for MALDI-TOF MASS + + ++ ++ + + + + p u ls e d U V o r IR la s e r ( 3 - 4 n s ) d e te c to r v a c u u m s t r o n g e le c tr ic fie ld T im e O f F lig h t t u b e p e p tid e m ix tu r e e m b e d d e d in lig h t a b s o r b in g c h e m ic a ls ( m a tr ix ) c lo u d o f p r o to n a te d p e p tid e m o le c u le sa c cV
  • 61.
    Principal for MALDI-TOFMASSPrincipal for MALDI-TOF MASS L in e a r T i m e O f F li g h t t u b e R e f le c t o r T im e O f F l ig h t t u b e d e t e c t o r r e f le c t o r io n s o u r c e io n s o u r c e d e t e c t o r t im e o f flig h t t im e o f flig h t
  • 62.
    MALDIMALDI == SELDISELDI 337nm UV laser MALDI cyano-hydroxy cinnaminic acid
  • 63.
  • 64.
    Background of fragmentation •The impact of a stream of high energy electrons causes the molecule to lose an electron forming a radical cation. – A species with a positive charge and one unpaired electron + e - C H H H H H H H HC + 2 e - Molecular ion (M+ ) m/z = 16
  • 65.
    Background • The impactof the stream of high energy electrons can also break the molecule or the radical cation into fragments. (not detected by MS) m/z = 29 molecular ion (M + ) m/z = 30 + C H H H + H HH C H H C H H H C H H C H H H C H H + e - H C H H C H H H m/z = 15
  • 66.
    Background • Molecular ion(parent ion): – The radical cation corresponding to the mass of the original molecule • The molecular ion is usually the highest mass in the spectrum – Some exceptions w/specific isotopes – Some molecular ion peaks are absent. H H H HC H C H H C H H H
  • 67.
    Background • Mass spectrumof ethanol (MW = 46) M+
  • 68.
    Background • The cationsthat are formed are separated by magnetic deflection.
  • 69.
    Background • Only cationsare detected. – Radicals are “invisible” in MS. • The amount of deflection observed depends on the mass to charge ratio (m/z). – Most cations formed have a charge of +1 so the amount of deflection observed is usually dependent on the mass of the ion.
  • 70.
    Background • The resultingmass spectrum is a graph of the mass of each cation vs. its relative abundance. • The peaks are assigned an abundance as a percentage of the base peak. – the most intense peak in the spectrum • The base peak is not necessarily the same as the parent ion peak.
  • 71.
  • 72.
    Background • Most elementsoccur naturally as a mixture of isotopes. – The presence of significant amounts of heavier isotopes leads to small peaks that have masses that are higher than the parent ion peak. • M+1 = a peak that is one mass unit higher than M+ • M+2 = a peak that is two mass units higher than M+
  • 73.
    Easily Recognized Elementsin MS • Nitrogen: – Odd number of N = odd MW CH3CN M + = 41 SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/2/09)
  • 74.
    Easily Recognized Elementsin MS  Bromine:  M+ ~ M+2 (50.5% 79 Br/49.5% 81 Br) 2-bromopropane M+ ~ M+2 SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/1/09)
  • 75.
    Easily Recognized Elementsin MS • Chlorine: – M+2 is ~ 1/3 as large as M+ Cl SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/2/09) M+2 M+
  • 76.
    • Sulfur: – M+2larger than usual (4% of M+ ) Easily Recognized Elements in MS M+ Unusually large M+2 S SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/1/09)
  • 77.
    Easily Recognized Elementsin MS • Iodine – I+ at 127 – Large gap Large gap I+ M+ SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/2/09) ICH2CN
  • 78.
    Fragmentation Patterns • Theimpact of the stream of high energy electrons often breaks the molecule into fragments, commonly a cation and a radical. – Bonds break to give the most stable cation. – Stability of the radical is less important.
  • 79.
    Fragmentation Patterns • Alkanes –Fragmentation often splits off simple alkyl groups: • Loss of methyl M+ - 15 • Loss of ethyl M+ - 29 • Loss of propyl M+ - 43 • Loss of butyl M+ - 57 – Branched alkanes tend to fragment forming the most stable carbocations.
  • 80.
    Fragmentation Patterns • Massspectrum of 2-methylpentane
  • 81.
    Fragmentation Patterns • Alkenes: –Fragmentation typically forms resonance stabilized allylic carbocations
  • 82.
    Fragmentation Patterns • Aromatics: –Fragment at the benzylic carbon, forming a resonance stabilized benzylic carbocation (which rearranges to the tropylium ion) M+ CH H CH Br H C H H or
  • 83.
    Fragmentation Patterns • Alcohols –Fragment easily resulting in very small or missing parent ion peak – May lose hydroxyl radical or water • M+ - 17 or M+ - 18 – Commonly lose an alkyl group attached to the carbinol carbon forming an oxonium ion. • 1o alcohol usually has prominent peak at m/z = 31 corresponding to H2C=OH+
  • 84.
    Fragmentation Patterns • MSfor 1-propanol M+ M+ -18 CH3CH2CH2OH H2C OH SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/28/09)
  • 85.
    Fragmentation Patterns • Amines –Odd M+ (assuming an odd number of nitrogens are present) – α-cleavage dominates forming an iminium ion CH3CH2 CH2 N H CH2 CH2CH2CH3 CH3CH2CH2N CH2 H m/z =72 iminium ion
  • 86.
  • 87.
    Fragmentation Patterns • Ethers –α-cleavage forming oxonium ion – Loss of alkyl group forming oxonium ion – Loss of alkyl group forming a carbocation
  • 88.
    Fragmentation Patterns H OCHCH3 MS of diethylether (CH3CH2OCH2CH3) CH3CH2O CH2 H O CH2
  • 89.
    Fragmentation Patterns • Theimpact of the stream of high energy electrons often breaks the molecule into fragments, commonly a cation and a radical. – Bonds break to give the most stable cation. – Stability of the radical is less important.
  • 90.
    Fragmentation Patterns • Alkanes –Fragmentation often splits off simple alkyl groups: • Loss of methyl M+ - 15 • Loss of ethyl M+ - 29 • Loss of propyl M+ - 43 • Loss of butyl M+ - 57 – Branched alkanes tend to fragment forming the most stable carbocations.
  • 91.
    Fragmentation Patterns • Massspectrum of 2-methylpentane
  • 92.
    Fragmentation Patterns • Alkenes: –Fragmentation typically forms resonance stabilized allylic carbocations
  • 93.
    Fragmentation Patterns • Aromatics: –Fragment at the benzylic carbon, forming a resonance stabilized benzylic carbocation (which rearranges to the tropylium ion) M+ CH H CH Br H C H H or
  • 94.
    Fragmentation Patterns Aromatics mayalso have a peak at m/z = 77 for the benzene ring. NO2 77 M+ = 123 77
  • 95.
    Fragmentation Patterns • Alcohols –Fragment easily resulting in very small or missing parent ion peak – May lose hydroxyl radical or water • M+ - 17 or M+ - 18 – Commonly lose an alkyl group attached to the carbinol carbon forming an oxonium ion. • 1o alcohol usually has prominent peak at m/z = 31 corresponding to H2C=OH+
  • 96.
    Fragmentation Patterns • MSfor 1-propanol M+ M+ -18 CH3CH2CH2OH H2C OH SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/28/09)
  • 97.
    Fragmentation Patterns • Amines –Odd M+ (assuming an odd number of nitrogens are present) – α-cleavage dominates forming an iminium ion CH3CH2 CH2 N H CH2 CH2CH2CH3 CH3CH2CH2N CH2 H m/z =72 iminium ion
  • 98.
  • 99.
    Fragmentation Patterns • Ethers –α-cleavage forming oxonium ion – Loss of alkyl group forming oxonium ion – Loss of alkyl group forming a carbocation
  • 100.
    Fragmentation Patterns • Aldehydes(RCHO) – Fragmentation may form acylium ion – Common fragments: • M+ - 1 for • M+ - 29 for RC O R (i.e. RCHO - CHO) RC O
  • 101.
    Fragmentation Patterns • MSfor hydrocinnamaldehyde M+ = 134 C C C H H H H H O 133 105 91 105 91 SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/28/09)
  • 102.
    Fragmentation Patterns • Ketones –Fragmentation leads to formation of acylium ion: • Loss of R forming • Loss of R’ forming RC O R'C O RCR' O
  • 103.
    Fragmentation Patterns • MSfor 2-pentanone CH3CCH2CH2CH3 O M+ CH3CH2CH2C O CH3C O SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/28/09)
  • 104.
    Fragmentation Patterns • Esters(RCO2R’) – Common fragmentation patterns include: • Loss of OR’ – peak at M+ - OR’ • Loss of R’ – peak at M+ - R’
  • 105.
    Frgamentation Patterns M+ = 136 C O OCH3 105 77 105 77 SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/28/09)
  • 106.
    Rule of Thirteen •The “Rule of Thirteen” can be used to identify possible molecular formulas for an unknown hydrocarbon, CnHm. – Step 1: n = M+ /13 (integer only, use remainder in step 2) – Step 2: m = n + remainder from step 1
  • 107.
    Rule of Thirteen •Example: The formula for a hydrocarbon with M+ =106 can be found: – Step 1: n = 106/13 = 8 (R = 2) – Step 2: m = 8 + 2 = 10 – Formula: C8H10
  • 108.
    Rule of Thirteen •If a heteroatom is present, – Subtract the mass of each heteroatom from the MW – Calculate the formula for the corresponding hydrocarbon – Add the heteroatoms to the formula
  • 109.
    Rule of Thirteen Example:A compound with a molecular ion peak at m/z = 102 has a strong peak at 1739 cm-1 in its IR spectrum. Determine its molecular formula.
  • 110.
    References • Chatwal GR,Anand SK. Instrumental method of chemical analysis, Himalaya publishing house. • Sharma YR. Elementary organic spectroscopy. • Willard,merritt,dean. Instumental methods of analysis. • www.google.com/images 06/13/15 110
  • 111.