Mass Spectroscopy Ppt 5w5u11

MAEERS MAHARASHTRA INSTITUTE OF PHARMACY, PUNE

A SEMINAR ON MASS SPECTROMETRY As per Pune University syllabus 1st semester

Presented By: Mr. Patil Somnath Ashok Guided By: Principal Dr. B.S. Kuchekar 1

CONTENTS: • Introduction • Definition • Classification Atomic MS Molecular MS • Principle of MS • Instrumentation Ion sources Mass analyzers Detectors Vacuum system • Applications

2

Introduction: • In Mass Spectroscopy (MS), atomic and molecular weights are generally expressed in of atomic mass units (amu). The atomic mass unit is based upon a relative scale in which the reference is the carbon isotope 12C , which is assigned a mass of exactly 12 6 amu. Thus the amu is defined as 1/12 of the mass of one neutral carbon atom. 3

Mass Spectroscopy • Mass spectroscopy is perhaps one of the most widely applicable of all the analytical tools available to the analytical chemist in the sense that this technique is capable of providing information about :

4

• the qualitative and quantitative composition of both organic and inorganic analytes in complex mixtures • this instrument measures compounds with molecular masses up to 200, 000 Daltons. • the structures of a wide variety of complex molecular species • isotopic ratios of atoms in samples and • the structure and composition of solid surfaces. 5

Mass Spectroscopy

Molecular Mass

Spectroscopy

Atomic Mass

Spectroscopy

6

Atomic Mass Spectrometry • Nearly all elements in the periodic table can be determined by mass spectrometry • More selective and sensitive than optical instruments • Simple spectra • Isotope ratios • Much more expensive instrumentation 7

Atomic Mass Spectrometry (Inorganic MS) • Mass Spectrometers • I-MS

• Spark Source MS • Glow-Discharge MS

• Elemental Surface Analysis by MS • Laser Ablation I-MS 8

Atomic Mass Spectrometry processes: • Atomization (sample intro) • Conversion to ions • Separation based on m/z ratio

• Detection In other forms of MS (GC-MS or MS of organic compounds), sample introduction does not involve making atoms, just getting molecules into the high vacuum system 9

Advantages of Atomic Mass Spectrometry over Optical Atomic Spectrometry: 1) Detection limits are better, sometimes several orders of magnitude 2) Very simple spectra 3) Ability to measure isotope ratios

Disadvantages: 1) Equipment cost 2) Instrument drift 3) Isotopic interferences 10

Advances In Atomic Mass Spectrometry

11

Molecular Mass Spectroscopy

12

Principle • Mol. Are bombarded with a beam of energetic electron

• Ionization of mol. & fragmentation • Some of which are +ve • Each ion has a particular ratio of mass to charge i.e. m/e ratio • For most of ion charge is 1 • So m/e =mol. Wt of ion.

13

Mass Spectrometry Simplest form of mass spectrometer performs 4 essential functions: (under vacuum 10-6 mm Hg)

• It subjects vaporized molecules to bombardment by a stream of high-energy electrons, converting these molecules to ions • These ions are then accelerated in an electric field • The accelerated ions are then separated according to their mass-to-charge ratio in a magnetic or electric field • The ions that have a particular mass-to-charge ratio are then detected by a device that counts the number of ions striking it. 14

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

15

INSTRUMENTATION: 10-5 to 10-8 Torr System Inlet

Ion Source

Mass Analyzer

Vacuum System

Detector

Signal processor

MASS SPECTROMETER m/z 16

Instrumentation available: • VG Analytical 70-250-SE

Normal geometry double focusing MS.

• Micromass TofSpec2E

Reflectron MALDI-TOFMS.

• ThermoQuest TraceMS

Single quadrupole GC-MS.

• Bruker ApexIII

FT-ICR-MS.

• ThermoBioAnalysis Dynamo

Linear MALDI-TOFMS.

• Micromass Platform II

Open access single quadrupole MS.

• Waters ZMD

Open access single quadrupole MS.

• ThermoQuest TraceMS

Open access single quadrupole GC-MS.

• ThermoFinnigan LCQ

Ion trap.

• Micromass Platform II

Single quadrupole MS

17

Resolution & 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 M • Resolving power is defined as:

∆M • M is the mass number of the observed mass (DM) is the difference between two masses that can be separated 18

e.g. the mass spectrum of HCl 100

36.5

%

0 mass 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 19

Scan EI+ 7.58e12

36.0

100

%

38.0

0 25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

mass 49

20

1. Sample Introduction: • All Glass Heated Inlet System (AGHIS): • Direct Insertion Probe (DIP):  MALDI pattern

• Direct Inlets:  Gas chromatography column  API interface  GC, HPLC, 21

The Sample Inlet System

• Batch Inlet Systems: • simplest and involve volatilization of liquid • For gases, introduced into the metering volume container and expanded into the reservoir flask

•The Direct Probe Inlet: • used for solids and non-

volatile liquids

•sample is introduced by means of a sample holder, or probe, • the amount of the sample to be analyzed is small.

22

2. IONISATION SOURCES CLASSIFICATION:

GAS PHASE ION SOURCE: DESORPTION ION SOURCE:

HARD SOURCE: SOFT SOURCE:

23

1. Gas Phase: 1. Electron ionization (EI) 2. Chemical ionization (CI) 3. Field Ionization (FI)

2. Desorption 1. 2. 3. 4. 5. 6. 7. 8.

Field Desorption (FD) Fast Atom Bombardment (FAB) Secondary Ion Mass Spectrometry (SIMS) Laser Desorption (LD) Plasma Desorption (PD) Thermal Desorption Thermo spray ionization (TS) Electrospray (ES) 24

Gas-Phase Sources Gas-phase sources require volatilization of the sample before ionization and thus are limited to thermally stable compounds that have boiling points less than about 500°C.

25

Ion sources for gases:

ELECTRON-IMPACT SOURCE

26

• Electron Impact Source :

In the sources,

electrons emitted from a filament are accelerated by a potential of about 70 V and made to collide with gaseous atoms or molecules of the sample

causing ionization. Electron-impact ionization is not very efficient and only about one molecule in a million undergoes the primary reaction

M + e- = M.+ + 2e27

Electron Impact Advantages • • • • •

Well-Established Fragmentation Libraries Insoluble Samples Interface to GC Non-Polar Samples

Disadvantages • • • • •

Parent Identification Need Volatile Sample Need Thermal Stability No Interface to LC Low Mass Compounds (<1000 amu) • Solids Probe Requires Skilled Operator

28

Chemical ionization: Transfers less energy, produces less fragmentation Introduce small molecule e.g. CH4, NH3, C4H10 into closed ion source, 1 Torr, as reagent, R, to produce electronimpact ionization. R is selective.

e.g. E.I.: CH4 + e  CH3+, CH4+…+ 2e then 2ary ions: CH4+ + CH4  CH5+ + CH3

or CH3+ + CH4  C2H5+ + H2 (i.e. large amount of gas R, small amount of sample)

29

Behavior of R : 1. As strong acid (proton transfer): e.g. AH + CH5+ → AH2+ + CH4 



analyte protonated

2. Hydride ion transfer (H- abstraction): C2H5+ + AH → A+ + C2H6  fragmentation less extensive than with E. I.

3

. Charge transfer:

CH4+ + AH → CH4 + AH+ 30

Main differences C. I. & E.I. 1. C.I. reagent gas produces an ion that protonates analyte

M  MH+; AH  AH2+ These ions, with paired e, more stable than radical species from E.I

2. Pattern of fragmentation in C.I. is complementary to that in E.I. C.I. No molecular ion, but MH+. Simple spectra. Easy to identify mixtures.

31

3. Degree of fragmentation in C.I. (F.I.) smaller. •

M.S. more easily interpreted. E.I. wealth of fragments at expense of M+.

4. C.I. more sensitive than E.I.

•

Ionization concentrated in few ions. For E.I. ionization efficiency is 0.1%.

•

Residence time inc. & pressure of R inc. give more ions in C.I. 32

Chemical Ionization Advantages • Parent Ion • Interface to GC • Insoluble Samples

Disadvantages • • • • •

No Fragment Library Need Volatile Sample Need Thermal Stability Quantitation Difficult Low Mass Compounds (<1000 amu) • Solids Probe Requires Skilled Operator 33

Field ionization source:

34

1. Activate wire anode with sharp points, whiskers, 10-102 nm radius, by filling chamber with C6H5CN at 0.04-0.1 Torr, apply 104 V for 2h. (or just use sharp-pointed metal anode). 2. Introduce sample Some deposits on anode tip.

3. Anode is gun input, 105 V/cm between anode & cathode, under high vac. 4. Electric field sufficient to remove e-, but not excite M+ Little fragmentn (no e- collisions; ions move into analyzer in 10-6, 10-7 s). M+ and (M+1)+ abundant. About 10× less sensitive than E.I.

35

36

• The matrix chromospheres absorbs and distribute the energy of a laser, produced a plasma, • vaporizes and ionize the sample. • + rapid, convenient for molecular weight (singly charged ions mostly) • - MS/MS difficult, almost not compatible with LC coupling • <500’000Dac • Matrix materials used are: benzoic acid der. , cinnamic acid der. & nicotinic acid der.s 37

MALDI Advantages • Parent Ion • High Mass Compounds (>100,000 amu) • Thermally Labile Compounds (R.T.) • Easy to Operate

Disadvantages • No Fragment Library • Wide variety of matrices • Quantitation Difficult

38

2.Fast Atom Bombardment (FAB) The sample is dissolved in a liquid matrix such as glycerol, thioglycerol, m-nitro benzyl alcohol, or diethanolamine and a small amount (about 1 micro liter) is placed on a target.

The target is bombarded with a fast atom beam (for example, 6 keV xenon atoms) that desorbs molecular-like ions and fragments from the analyte. Xe Xe+

Xe + Xe+

e-

Xe+ + Xe

Is accelerated to ~6-10 keV, and through Xe

Xe

(Xenon with Kinetic energy) FAB

Cluster ions from the liquid matrix are also desorbed and produce a chemical background that varies with the matrix used. 39

Benefits: Sample introduction can be through direct insertion probe or LC/MS (continuous-flow FAB).

•

rapid, simple

•

relatively tolerant of variations in sampling

•

good for a large variety of compounds

•

Useful fragmentation pattern

• strong ion currents -- good for high-resolution measurements 40

Limitations: • high chemical background defines detection limits • may be difficult to distinguish low-molecular-weight compounds from chemical background • analyte must be soluble in the liquid matrix • no good for multiply charged compounds with more than 2 charges • requires a high concentration of the organic liquid matrix (typically 80 to 95% glycerol) which limits sensitivity Mass range Moderate Typically ~300 Da to about 6000 Da. 41

3. Thermal desorption Coat sample on thin wire insertion probe. Heat 100-200 oC s-1 to get desorption/evapn rather than decompn. Then use EI, CI etc. Use for inorganic compounds with low I.E. 3-6 eV.

4. Field desorption Coat field ionizn. emitter (carbon needle) with nonvolatile sample. Heat wire & apply high electric p.d. Useful for studying surface phenomena such as adsorbed or trapped species.

5.

252-

D set t=0

Plasma desorption

~m/2

252Cf

~m/2

~75MeV charge < 20

sample film on metal foil

Low ion yield, long counting time, but up to 20000amu.

10-50 cm to minimize fragmentation electronic excitation accelerates localized plasma of molecule & fragment ions

charged grid D

calibration m/z from t for Na+ 42

Deposit sample on foil and bombard with fission fragments of MeV energy.

6. ESI: sample solution sprayed across high p.d. from a needle into an interfaced orifice. Heat and flow gas desolvate the charged droplets, producing multiplycharged ions. Used for charged, polar or basic compds. up to 50 kDa. LC

N2(g)

to ms

+

to high vacuum pump: 10-4 -10-5 mbar

nebulizing gas

to rotary pump:

1 mbar

43

ESI Advantages

Disadvantages

• Parent Ion • High Mass Compounds (>100,000 amu) • Thermally Labile Compounds (<0º C) • Easy to Operate • Interface to HPLC • Zeptomole sensitivity with nanospray

• No Fragmentation • Need Polar Sample • Need Solubility in Polar Solvent (MeOH, ACN, H2O, Acetone are best) • Sensitive to Salts • Suppression

44

Types of ions produced in MS • • • • • •

Molecular ion/Parent ion Daughter ion Rearrangement ion Multiple charged ion Negative ion Metastable ion

45

Components of MS: 1.Mass Analyzers Mass Analyzer

1. 2. 3. 4. 5. 6.

Dispersion of the ions is based on mass-to-charge ratio

There are several type of mass analyzers.

Magnetic sector Electrostatic and Magnetic sector Quadrupole MS filter Ion trap analyzers TOF – Time-of-Flight FT-MS 46

2. Detector: Detector

Convert the beam of ions in an electrical signal that can be processed, stored, displayed and recorded in many ways.

Other:

Electron Multiplier (most commonly used) Faraday cup Photographic plates Scintillation type Vacuum System

3.Vacuum: MS require the high vacuum is maintained in all spectrometer components (except signal processing)

47

Vacuum Systems • Reduce gas molecules in analysis chamber – Ion detectors need low pressure environment – Don’t want to detect gas in system – Want to detect analyte before it can collide or react with gas in system – Surface analysis: a need for analyzing an unoxidized surface 48

Vacuum Pump Selection • Atmosphere to 10-2 Torr – Roughing pumps

• 10-4 – 10-8 Torr – Diffusion pumps – Turbo molecular pumps – Cryogenic pumps

• 10-9 – 10-12 Torr – Ion pumps 49

Vacuum Regions • Rough Vacuum – 760-1 Torr

• Mid Vacuum – 1 – 10-3 Torr

• High Vacuum – 10-3 – 10-7 Torr

• Ultrahigh Vacuum (UHV) – <10-7 Torr 50

Magnetic Sector Analyzers:

51

• Magnetic sector analyzers employ a permanent magnet or electromagnet to cause the beam from the ion source to travel in a circular path of 180, 90, or 60 degrees. Here, ions are formed by electron impact. • The translational energy of an ion of mass m and charge z upon exciting slit B is given by K = ZeV = ½ mv2 • where V is the voltage between A and B, v is the velocity of the ion after acceleration, and e is the charge of the ion. • The path in the sector described by the ions of a given mass and charge represents a balance between two forces acting upon them. These two forces are the centripetal force and the magnetic force and equating these two forces yields: • Bzev = mv2/r which rearranges to v = Bzer/m • Substituting the above equation into (1) gives m/z = B2r2e/2V 52

Double Focusing Instruments:

53

• These type of instruments, unlike singlefocusing which simply minimize directional errors, are designed to limit both the errors introduced because ions are initially moving in different directions and also the errors introduced due to the fact that ions of the same mass-to-charge ratio may have different translational energies. 54

The characteristics are listed below: • Classical shaped mass spectra • Very high reproducibility • Best quantitative performance of all mass spectrometer analyzers • High resolution • High sensitivity • High dynamic range

• Linked scan MS/MS does not need another analyzer • High energy CID (collision induced decay) MS/MS spectra are very reproducible

55

The limitations of sector instruments can be summarized as: • Not well-suited for pulsed ionization methods (e.g. MALDI) • Usually larger and higher costs both in purchase and

maintenance than other mass analyzers • Linked scanning MS/MS gives either limited precursor

selectivity with unit product-ion resolution or nominal precursor selection with poor product-ion resolution 56

Applications: • All organic MS analysis methods • Accurate mass measurements/peak-matching • Quantitation • Isotope ratio measurements

57

Time-of-Flight analyzer (TOF)

58

In TOF instruments, positive ions are produced periodically using

brief pulses of electrons, secondary ions or laser generated photons These pulses have typically a frequency of 10-50 kHz and lifetime 0.25ms

The ions produced are then accelerated by electric field pulse (103-104 V) that has the same frequency as ionization pulse but lags behind. The accelerated ions then into a field-free drift tube (about 1 m long) 59

Time-of-Flight (TOF) Advantages • Extremely High Mass Range (>1 MDa) • Fast Scanning

Disadvantages • Low Resolution (4000) • Low Accuracy (>200ppm) • MS/MS not possible

60

Quadrupole Mass Analysers:

61

62

63

The ion trap functions both as an analyser and an ion storage device. The ions are produced or introduced into the ion trap, trapped and can be progressively ejected form the ion trap volume by ramping of the rf voltage. The trapped ion can also be induced to undergo dissociation and hence produce the MSMS spectrum within the one analyser. Note the difference with FT-ICR-MS where the

frequency of the circulating ions is measured and Fourier Transformed in give the MS. 64

Reflectron Time-of-Flight (TOF)

65

Reflectron Time-of-Flight (TOF) Advantages

Disadvantages

• High Resolution (>20,000 in some models) • High Accuracy (<5ppm) • 10,000 Mass Range • Fast Scanning

• Low Resolution for MS/MS (PSD)

66

FT-ICR-MS

67

FT-ICR Fourier-transform ion cyclotron resonance

• Uses powerful magnet (5-10 Tesla) to create a miniature cyclotron • Originally developed in Canada (UBC) by A.G. Marshal in 1974 • FT approach allows many ion masses to be determined simultaneously (efficient)

• Has higher mass resolution than any other MS analyzer available

68

• Detectors: • Two common types of detectors: 1) Faraday Cup 2) Electron Multiplier

Faraday Cup: •

Ions are accelerated toward a grounded “collector electrode” As ions strike the surface, electrons flow to neutralize charge, producing a small current that can be externally amplified. Size of this current is related to the number of ions .

•

less sensitive 69

70

• Electron Multiplier: • Analogous to PMT • Durable, applicable to most analyzers • Ions strike surface of dynode • Generate electrons • Ejected electrons are • accelerated to other dynodes • Current is related to number of ions. 71

APPLICATIONS: 1. Elucidation of the structure of organic and biological molecules. 2. Determination of molecular mass of peptide, proteins and oligonucleotides. 3. Identification of components in thin layer and paper chromatograms. 4. Determination of amino acids sequences in sample of polypeptides and proteins. 5. Detection and identification of species separated by chromatography. 72

6. Identification of drugs of abuse and metabolites of drugs of abuse in blood, urine, and saliva. 7. Monitoring gases in patient’s breath during surgery. 8. Testing for the presence of drugs in blood in race horses and in Olympic athletes. 9. Analyses of aerosol particles. 10. Determination of pesticide residues in food. 11. Monitoring volatile organic species in water supplies.

73

Conclusions • Mass spectrometers exist in many different configurations to allow different problems to be solved • All mass spectrometers have a common architecture and relatively similar operating principles • Understanding the applications and limitations of MS in proteomics will help in understanding and meeting the bioinformatics needs in proteomics 74

Bibliography: • Instrumental Analysis By Skoog, Holler, Crouch 8th Indian Reprint 2010 Page No. 318 & 606 • Instrumental Methods Of Chemical Analysis By G.R. Chatwal & S.K. Anand 1st Edition 1979 Page No. 2.272 • Introduction to spectroscopy by Donald L. Pavia Gary M. Lampman George S. Kriz 3rd edition page no. 390 • www.studytemple.com • • • • • •

http://www.spectorscopymag.com/spectrbl.htm http://www.organicchemistryonline.com/OCOL.HTM# http://www.scimedi.com/chem-ed/analytic/ac-meths.htm http://masspecscripps.edu/hist.htm http://www.hdscience.com/ http://www.scimedia.com/chem-ed/ms/ms-intro.htm 75

76