Mass spectrometry - A detailed study on components

A detailed presentation on Mass spectrometry.

1. MASS SPECTROMETRY Dr. P.Samuel Assistant Professor of Biotechnology Ayya Nadar Janaki Ammal College (Autonomous) Sivakasi

2. MASS SPECTROMETRY Definition: Mass spectrometry  ( MS ) is an analytical technique that ionizes  complex chemical species (Proteins)  and sorts the  ions  based on their  mass-to-charge ratio . In simpler terms, a  mass spectrum  measures the masses within a sample. Mass spectrometry is used in many different fields and is applied to pure samples as well as complex mixtures.

3. What is a spectrometry? Spectrophotometry  is a method to measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through sample solution. The basic principle is that each compound absorbs or transmits light over a certain range of wavelength.

4. What is the difference between spectrometry and spectroscopy? The term  spectroscopy  is normally reserved for measurements of the electromagnetic spectrum. Words ending in - scopy mean "looking at" whereas words in - metry mean "measurement of". In practice, the two are used interchangeably, with the exception of mass  spectrometry  where the term "mass  spectroscopy " is discouraged.

5. When was the mass spectrometer invented? The  mass spectrometer  was  invented  by F. W. Aston shortly after World War I. By 1927 Aston had built an instrument that was accurate to more than 1 part in 10,000, and  mass spectrometry  became the method of choice for measuring the relative mass  of an atom or molecule.

6. What does a mass spectrometer tell you? A  mass spectrometer  produces charged particles (ions) from the chemical substances that are to be analyzed. The  mass spectrometer  then uses electric and magnetic fields to measure the  mass  ("weight") of the charged particles.

7. What is the principle of the mass spectrometer? Basic Principle . A   mass spectrometer  generates multiple ions from the sample under investigation, it then separates them according to their specific  mass -to-charge ratio (m/z), and then records the relative abundance of each ion type. ... This molecular ion undergoes fragmentation.

8. History and Development of MS Year Contribution 1886 Eugen Goldstein  observes  canal rays . 1898 Wilhelm Wien  demonstrates that canal rays can be deflected using strong electric and magnetic fields. He shows that the  mass-to-charge ratio  of the particles have opposite polarity and is much larger compared to the electron. He also realizes that the particle mass is similar to the one of hydrogen particle. 1898 J. J. Thomson  measures the  mass-to-charge ratio  of electrons. 1901 Walter Kaufmann  uses a mass spectrometer to measure the relativistic mass increase of electrons. 1905 J. J. Thomson  begins his study of positive rays. 1906 Thomson is awarded the Nobel Prize in Physics "in recognition of the great merits of his theoretical and experimental investigations on the conduction of electricity by gases"

9. Year Contribution 1913 Thomson is able to separate particles of different  mass-to-charge ratios . He separates the  20 Ne and the  22 Ne isotopes, and he correctly identifies the  m/z  = 11 signal as a doubly charged  22 Ne particle. 1919 Francis Aston  constructs the first velocity focusing mass spectrograph with mass resolving power of 130. 1922 Aston is awarded the Nobel Prize in chemistry "for his discovery, by means of his mass spectrograph, of isotopes, in a large number of non-radioactive elements, and for his enunciation of the whole-number rule." 1931 Ernest O. Lawrence  invents the  cyclotron . 1934 Josef Mattauch  and  Richard Herzog  develop the double-focusing mass spectrograph. 1936 Arthur J. Dempster  develops the  spark ionization  source. 1937 Aston constructs a mass spectrograph with resolving power of 2000. 1939 Lawrence receives the Nobel Prize in Physics for the cyclotron. 1942 Lawrence develops the  Calutron  for  uranium   isotope separation . 1943 Westinghouse markets its mass spectrometer and proclaims it to be "A New Electronic Method for fast, accurate gas analysis". 1946 William Stephens presents the concept of a  time-of-flight  mass spectrometer.

10. Year Contribution 1954 A. J. C. Nicholson (Australia) proposes a hydrogen transfer reaction that will come to be known as the  McLafferty rearrangement . 1959 Researchers at  Dow Chemical  interface a  gas chromatograph  to a mass spectrometer. 1964 British Mass Spectrometry Society established as first dedicated mass spectrometry society. It holds its first meeting in 1965 in London. 1966 F. H. Field and M. S. B. Munson develop  chemical ionization . 1968 Malcolm Dole  develops electrospray ionization. 1969 H. D. Beckey develops  field desorption . 1974 Comisarow and Marshall develop  Fourier Transform Ion Cyclotron Resonance  mass spectrometry. 1976 Ronald MacFarlane and co-workers develop  plasma desorption mass spectrometry . 1984 John Bennett Fenn  and co-workers use  electrospray  to ionize biomolecules. 1985 Franz Hillenkamp , Michael Karas and co-workers describe and coin the term  matrix-assisted laser desorption ionization  (MALDI).

11. Year Contribution 1987 Koichi Tanaka  uses the “ultra fine metal plus liquid matrix method” to ionize intact proteins. 1989 Wolfgang Paul  receives the Nobel Prize in Physics "for the development of the ion trap technique". 1999 Alexander Makarov presents the  Orbitrap  mass spectrometer. 2002 John Bennett Fenn and  Koichi Tanaka  are awarded one-quarter of the Nobel Prize in chemistry each "for the development of soft desorption ionisation methods ... for mass spectrometric analyses of biological macromolecules."

12. Components of MS

13. Mass spectrometry uses an instrument called a mass spectrometer. The  main components of a mass spectrometer are: Inlet system (LC, GC, Direct probe etc...) Ion source (EI, CI, ESI, APCI, MALDI, etc...) Mass analyzer (Quadrupole, TOF, Ion Trap, Magnetic Sector) Detector (Electron Multiplier, Micro Channel Plates MCPs)

14. Samples can be introduced to the mass spectrometer directly via solids probe, or in the case of mixtures, by the intermediary of chromatography device (e.g. Gas chromatography, Liquid chromatography, Capillary electrophoresis, etc...). Once in the source, sample molecules are subjected to ionization. Ions formed in the source (molecular and fragment ions) acquire some kinetic energy and leave the source. A calibrated analyzer then analyzes the passing ions as a function of their mass to charge ratios. Different kind of analyzer(s) can be used, Magnetic, Quadrupole, Ion trap, Fourier Transform, Time of Flight, etc...The ion beam exiting the analyzer assembly is then detected and the signal is registered.  Common ionization method acronyms include: EI=Electron Impact; CI=Chemical Ionization; SIMS=Secondary Ions Mass Spec; FAB=Fast Atom Bombardment;

15. Ionization Methods Selection of the proper ionization method for the analysis of your sample is extremely important.  Although we can offer suggestions, it is your responsibility to understand and select the method(s) appropriate for your research compounds .   Electron Impact EI Ionization Chemical Ionization CI Negative Ion Chemical Ionization Electrospray Ionization Techniques Matrix Assisted Lazer Desorption  Atmospheric Pressure Chemical Ionization APCI

16. Analysis of Ions It is possible to use several different physical parameters to achieve mass separation.   Common types of mass analyzers are discussed below.  Each has advantages and disadvantages. Magnetic Sector Mass Spectrometer

17. Ion source: There are several types of ionization methods in mass spectrometry. The physical basis of ionization methods are very complex and outside the scope of the course. Most common methods are: (a) Matrix-assisted laser desorption/ionization (MALDI) This method of ionization is a soft ionization method and results in minimum fragmentation of sample. This method is used for non-volatile, and thermally labile compounds such as proteins, oligonucleotides, synthetic polymers. Sample is mixed with 1000 times molar excess of sample and spotted onto a metal plate and dried. Matrix plays a key role in this technique by absorbing the laser light energy and causing a small part of the target substrate to vaporize. Although, the process of forming analyte ions is unclear, it is believed that matrix which has labile protons, such as carboxylic acids, protonates neutral analyte molecules after absorbing laser light energy.

18. (b) Electrospray Ionization (ESI) Electrospray Ionization (ESI) is a preferred method of ionization when the sample is in liquid form. This is also a soft method of ionization and results in less fragmentation. ESI is a very valuable method for analysis of biological samples. The method was developed by John Fenn and he shared 2002 Nobel prize in chemistry for this work. The analyte is introduced either from a syringe pump or as the eluent flow from liquid chromatography with a flow rate 1µl min-1 . The analyte solution passes through the Electrospray needle (Stainless steel capillary with 75-150 µm internal diameters) that has a high potential difference (with respect to the counter electrode) applied to it (typically in the range from 2.5 to 4 kV). This forces the spraying of charged droplets from the needle with a surface charge of the same polarity to the charge on the needle. As droplet moves towards counter electrode cone (which passes it to analyzer), solvent evaporation occurs and droplet shrinks until it reaches the point that the surface tension can no longer sustain the charge (the Rayleigh limit) and at that point droplets break. This produces smaller droplets and the process is repeated. Finally after all solvent evaporated, charge is passed on to analyte.