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Molecular Mass Spectrometry

Molecular Mass Spectrometry. Prof. N. Rama Rao Chalapathi Institute of Pharmaceutical sciences, Guntur nadendla2000@yahoo.co.in. 44amu. 44amu. +1. +2. =. =. 44. mass (amu or dalton). charge(+). =. =. 22. m. m. z. z. I. Introduction.

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Molecular Mass Spectrometry

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  1. Molecular Mass Spectrometry Prof. N. Rama Rao Chalapathi Institute of Pharmaceutical sciences, Guntur nadendla2000@yahoo.co.in

  2. 44amu 44amu +1 +2 = = 44 mass (amu or dalton) charge(+) = = 22 m m z z I. Introduction • Mass Spectrometry - used to determine the mass and structure of molecules based on the mass-to-charge (m/z) ratio of the molecular ion and its fragments. • m/z = mass-to-charge ratio = • Example: CH3-CH2-CH3 + e- CH3-CH2-CH3+ + 2e- CH3-CH2-CH3 + e- CH3-CH2-CH3++ + 3e-

  3. • Spectra are plotted in terms of relative molecular ion abundance vs. m/z. • Molecular ion peak (M) - the peak arising from the entire molecule with a +1 charge. • Base peak - the peak with the highest abundance (i.e. the most stable molecular fragment). • Abundance is commonly expressed as a percentage of the base peak, where the base peak = 100% abundance.

  4. • All mass spectrometers are constructed of the same basic components. • The inlet system, ion source, mass analyzer, and detector are incased in a vacuum chamber so air molecule (N2, O2, CO2, etc.) will not interfere with the analysis. • The ion source and the mass analyzer are the two most important components of any mass spectrometer.

  5. II. Ion Sources • A mass spectrometer works by using magnetic and electric fields to exert forces on • charged particles (ions) in a vacuum. Therefore, a compound must be charged or • ionized to be analyzed by a mass spectrometer. • Furthermore, the ions must be introduced in the gas phase into the vacuum system of • the mass spectrometer. • This is easily done for gaseous or heat-volatile samples. • However, many (thermally labile) analytes decompose upon heating. These • kinds of samples require either desorption or desolvation methods if they are to • be analyzed by mass spectrometry. • Although ionization and desorption/desolvation are usually separateprocesses, the term "ionization method" is commonly used to refer to both ionization and desorption (or desolvation) methods.

  6. II. Ion Sources (cont……) • The component responsible for the formation of the molecular ions. • There are a variety of techniques used to form the molecular ions and the appearance of the spectra is highly dependent upon the ion source. • There are two major categories of ion sources: A) Gas-Phase - samples are vaporized prior to ionization. This technique is useful for compounds with masses less than 1000 amu and boiling points below 500° C. B) Desorption - molecular ions are formed from solid or liquid samples. This technique is useful for nonvolatile and thermally unstable compounds.

  7. • Ion sources are also classified by the energetics of the ionization process: A) Hard Sources - impart sufficient energy on the analyte to leave the molecule in an excited state in which relaxation results in the rupture of bonds. Electron Impact Ionization A) Soft Sources - cause little fragmentation and the spectra have few, if any, peaks besides the M peak. Field Ionization

  8. • There are varying degrees of source “hardness”. Electron Impact Ionization (EI) Chemical Ionization (CI) Field Desorption (FD)

  9. A. Electron Impact (EI) Ionization • A hard, gas phase ion source. • The analyte molecules pass through a stream of electrons which bombard the molecule and dislodge an electron. M + e- M• + + 2e- • The positive ions are attracted to the accelerator plate by a potential ( 70 V) applied between the accelerator plate and the repeller. • Results in a highly excited M•+, which undergoes fragmentation and rearrangement.

  10. • The spectra from EI mass spectrometers have many peaks arising from many fragments (daughter peaks).

  11. • Isotope Peaks - peaks with a m/z greater than M in an electron impact mass spectrum are usually due to the natural abundance of isotopes. • The relative abundance of these peaks are useful in determining the carbon, oxygen, sulfur, bromine, and chlorine content of a compound being analyzed.

  12. • Advantages and disadvantages of electron impact ionization: Advantages: 1) Good sensitivity 2) The fragmentation pattern allows for unambiguous identification of an analyte. Disadvantages: 1) Samples must be volatilized, so the technique is not useful for high boiling or thermally unstable compounds. 2) Extensive fragmentation can cause the disappearance of the M peak.

  13. B. Chemical Ionization (CI) • Same setup as EI except the ionization chamber is pressurized with a reagent gas. • The reagent gas is present in a 103 to 104 excess over the analyte. • The reagent gas, usually methane, is preferentially ionized. CH4 + e- CH4•+ + 2e- • The primary ions CH4•+ and CH3+ form, which go on to give secondary ions. CH4•+ + CH4 CH5+ + CH3• CH3+ + CH4 C2H5+ + H2 CH4 + C2H5+ C3H5+ + 2H2 • Collisions between the reagent ions and the analyte cause proton transfer and hydride transfer to occur. CH5+ + MH  MH2+ +CH4 C2H5+ + MH  MH2+ + C2H4 C2H5+ + MH  M+ + C2H6 Proton Transfer Hydride Transfer • Spectra contain M+1 & M-1 molecular ion peaks.

  14. C. Desorption Techniques • Soft ionization techniques that usually result in spectra that consist of only M or M+1 peaks. • Commonly used for biological samples (i.e. proteins & DNA) or thermally unstable molecules and can measure molecular weights that exceed 10,000 amu. 1. Electrospray Ionization (ESI) • The most common ionization technique used to analyze biomolecules. • Can be used to analyze biological macromolecules > 100,000 amu. • The sample is pumped through a needle surrounded by several kV of potential. • The charged spray of ultra-fine droplets of sample then passes into a capillary. • While in the capillary the solvent evaporates and the charge is attached to the analyte.

  15. 2. Fast Atom Bombardment (FAB) • The sample is prepared in a glycerol matrix and bombarded with high velocity argon or xenon atoms. • Analyte anions and cations sputter off the sample, but only cations enter the mass analyzer due to a negatively charged accelerator/repeller plate at the analyzer inlet. • The matrix reduces fragmentation of the analyte by absorbing most of the vibrational energy imparted by the fast atom stream. • Used primarily for high molecular weight polar compounds.

  16. 3. Matrix Assisted Laser Desorption/Ionization (MALDI) • The sample is prepared in an aqueous/alcohol solution and mixed with a large excess of a radiation-absorbing matrix material. • The sample matrix is then dried (evaporated) on the surface of a metallic probe. • The mixture is then irradiated with a pulsed laser beam of the same wavelength that the radiation-absorbing matrix absorbs. Analyte cations are released from the mixture and enter a time-of-flight mass analyzer. • The entire mass spectrum is obtained between laser pulses. • MALDI has found widespread application for large (mw > 100,000) biological macromolecules since its inception in 1988.

  17. III. Mass Analyzers A) Resolution - measurement of a mass analyzers ability to separate masses that are very close together. R = resolution m = average mass of two adjacent peaks Dm= mass difference of two adjacent peaks • Two peaks are considered resolved if the height of the valley between the peaks is < 10% the height of the shortest peak. • Example: What is the resolution of a mass analyzer that resolves peaks at m/z = 436.036 and 436.055?

  18. B. Some Common Mass Analyzers 1. Magnetic Sector Mass Analyzer • Select mass based on centrifugal force in a magnetic field and the velocity of the molecular ion in the magnetic field. • Curvature of molecular ions flight path depends on the mass of the ion, velocity of the ion, and the magnetic field strength. • Mass is selected by varying either the strength of the magnetic field or the velocity of the ions. • The velocity of the ions can be varied by adjusting the potential difference between the repeller plate and the accelerator plates in an EI ionizer.

  19. 2. Quadrapole Filter • Constructed of four metal rods, which have an alternating charge applied. • At a given rate of alternation of the rod charges, only ions of the proper m/z will pass through the quadrapole and reach the detector slit. • Quadrapole filters have very fast scan times (< 1 s) and are often used as detectors for gas chromatography or liquid chromatography systems.

  20. 3. Time-of-Flight (TOF) Mass Analyzers • Select mass based on the time it takes an ion to fly through a field free, evacuated tube and reach a negatively charged detector. • Ions with higher m/z will take longer to reach the detector than ions of lower m/z. • Used for high molecular weight compound due to the limited resolution and sensitivity of the analyzer.

  21. 3. Ion Trap (Ion Cyclotron) • Constructed of a donut-shaped ring electrode and a second pair of electrodes that form end caps. • A current is placed on the ring electrode inducing a magnetic field in the trap. The gaseous ions that enter the trap will circulate in a plane perpendicular to the magnetic field, effectively trapping the molecular ions. • The potential on the end cap electrodes is then scanned. The ions will spin out of the trap to the detector as a function of the mass-to-charge ratio and the strength of the potential on the end caps. • Ion traps have a limited mass range (500 – 1000 amu) and selectivity. • Low cost alternative to a quadrapole filter detector for a gas chromatography system.

  22. Mass Spectrometry: Mass and Molecular Formula The Mass of a Charged Particle Can Be Measured In A Mass Spectrometer Sample Molecule Held in Gas Phase Radical Cations are High Energy Species and Are Capable of Undergoing Fragmentation

  23. Mass Spectrometer - General Layout Typical Mass Spectrum Only charged particles are deflected by the Magnet

  24. Mass Spectrum of Toluene BASE PEAK m/z = 91 C7H7+. PARENT ION: P m/z = 92 C7H8+. PEAKS DUE TO PARENT ION FRAGMENTATION: DAUGHTER IONS P+1 m/z = 93 12C613C1H8 i) The P+1 Peak is Approximately 1% of the Intensity of the Parent Ion. Why? ii) The Level of Fragmentation is Quite Low. Why?

  25. Fragmentation of Toluene Parent Ion C7H8+. m/z = 92 Benzyl cations are stabilized by resonance Positive charge smeared across 7 carbon atoms

  26. Different Molecules Can Have the Same Molecular Weight! In most cases we don’t know the formula of our molecule ahead of time……. SO How can we distinguish between A and B? A B

  27. Accurate Mass Measurement is the Solution! A B Mass Spectrometers are accurate enough to distinguish between molecules which have the same molecular formula

  28. Parent Ions Undergo Fragmentation Parent Peaks (M+.) Daughter Peaks Each Molecule Has a Unique Fragmentation Pattern

  29. Mass Spectrometer - Location of Fragmentation Fragmentation Occurs Here Typical Mass Spectrum Only charged particles are deflected by the Magnet

  30. The Course of Fragmentation is Directed by Daughter Ion Stability

  31. Fragmentation Patterns - Formation of Acylium Cations

  32. Fragmentation Patterns - Formation of Acylium Cations Base Peak C4H9+ m/z = 57 Parent Peak m/z = 142 C5H9O+ m/z = 85

  33. The Course of Fragmentation is Directed by Daughter Ion Stability (Alkenes) m/z = 72 Remember 3o > 2o >> 1o cations No Parent Peak at m/z = 72! WHY?

  34. Fragmentation Patterns - Elimination of Water No Parent Peak at m/z = 74! WHY?a E1 Elimination Reactions

  35. Fragmentation Patterns - McLafferty Rearrangement

  36. McLafferty Rearrangement of Butyraldehyde C3H4O, m/z = 44 C4H8O, m/z = 72 M +1 You can recognize a McLafferty Rearrangement by loss of 44 mass units (ethylene)

  37. Applications for Mass Spectrometry Technology biomedical research • Genomics (Genotype) • Genetic disease markers (e.g. SNP’s) • Proteomics (Phenotype) • Protein based disease markers • ‘Metabolomics’ (Chemotype) • Metabolite based disease markers • The ultimate expression of a disease • These 3 application • areas represent • new and exciting • opportunities for • mass spectrometry. • The 3 areas are • closely related to one • another and to • human health.

  38. Important Recent Developments in Biological Mass Spectrometry • API Ionization-MSMS (ITD or QqQ) • Electrospray • APCI • MALDI-TOF • Qq-TOF • FT-MS with MALDI and ESI

  39. Fastest Growing Applications • Biomedical • Proteomics • Genomics • Clinical “Metabolomics” (metabolic disorders, TDM) • Pharmaceutical • Preclinical pharmacology (Drug discovery) • Combinatorial chemistry (Drug discovery) • Clinical trials (Drug development)

  40. A A A A = Analyte; = Solvent; = Vacuum system Atmospheric Pressure Ionization (API) Before API e.g. GC/MS A A+ MS Liquid Introduction Mass Spectrometry After API e.g. LC/MS A A+ MS

  41. NH 2 Homocysteine Exact Mass: 135.04 Mol. Wt.: 135.18 Homocysteine by LC-MSMS HOOC SH Cardiovascular Risk Factor: mechanism currently unknown, however, believed will become as important to cardiovascular health and wellness as cholesterol Clinical Chemistry, 45(1999)1517

  42. Homocysteine: MSMS Product Ion Spectrum [M+H-HCO2H]+ [M+H]+

  43. 140+ 94+ 136+ 90+ Homocysteine in Plasma:LC-MSMS Response (15 mM)

  44. Homocysteine in Plasma:LC-MSMS Response (0.8 mM)

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