Mass Ionization Techniques

Introduction, Principle, Ion sources, Ionization techniques

1. IONIZATION TECHNIQUES IN MASS SPECTROMETRY Advanced instrumental analysis Presented by B. Likitha M. Pharmacy I-II Pharmaceutical analysis

2. INTRODUCTION Mass Spectrometry : Mass spectrometry is an analytical technique that involves the study in the gas phase of ionized molecules with the aim of one or more of the following: Molecular weight determination Structural characterization Gas phase reactivity study Qualitative and quantitative analysis of components in a mixture.

3. Basic Principle: Conversion of the sample into rapidly moving gaseous positive ions by electron bombardment and charged particles are separated according to their masses with or without fragmentation, which are then characterized by their mass to charge ratios (m/e) and relative abundances (mass spectrum) is the basic principle of mass spectrometry.

4. Working Principle: In this technique, molecules are bombarded with a beam of energetic electrons. The molecules are ionized and broken up into many fragments, some of which are positive ions. Each kind of ion has a particular ratio of mass to charge, i.e., m/e ratio. For most ions, the charge is one and thus, m/e ratio is simply the molecular mass of the ion. The ion pass through magnetic and electric fields to reach detector where they are detected and signals are recorded to give a mass spectra.

5. INSTRUMENTATION OF MASS SPECTROMETRY MASS SPECTRUM

6. CATEGORIES OF ION SOURCES ION SOURCES GAS PHASE SOURCES DESORPTION SOURCES Electron Impact Ionization (EI) Chemical Ionization (CI) Field Ionization (FI) Field Desorption (FD) Electrospray Ionization (ESI) Matrix Assisted Laser Desorption/Ionization (MALDI) Plasma Desorption (PD) Fast Atom Bombardment (FAB) Thermo spray Ionization (TI)

7. IONIZATION TECHNIQUES Electron Spray Ionization(ESI) Atmospheric Pressure Chemical Ionization(APCI) Atmospheric Pressure Photo Ionization(APPI) Matrix Assisted Laser Desorption Ionization Technique(MALDI) Fast Atom Bombardment(FAB)

8. Electrospray ionization The method generates ions from solution of a sample by creating fine spray of charged droplets. A solution of sample is pumped through a fine, charged stainless steel capillary needle at a rate of few micro litres /minute. The needle is maintained at a high electric field (several kilovolts) with respect to cylindrical electrode. The liquid phase pushes itself out of the capillary as a mist or aerosol of fine charged droplets. Set of aerosol droplets is produced by a process involving formation of a Taylor cone and a jet from the tip of this cone. These charged droplets are then passed through desolvating capillary where the solvent is evaporated in the vacuum and attachment of charge to the analyte molecules takes place.

9. Desolvating capillary uses warm nitrogen as nebulizing gas. The desolvating capillary is maintained under high pressure. As the droplets evaporate the analyte molecules comes closer together. These molecules become unstable as the similarly charged molecules comes closer together and the droplets explode once again. This is referred as Coulombic fission. The process repeats itself until the analyte is free from solvent and is lone ion. The ion then moves to the mass analyzer. In this method quassi -molecular ions are produced by addition of a proton (hydrogen ion) to give (M+H) or other cations such as sodium ion ( M+Na ) + or removal of hydrogen ion (M-H). Multiply charged ions are often observed and these ions are even electron species indicating that electrons have neither been added nor removed.

10. ELECTRON SPRAY IONIZATION Advantages: Used for analysis of high molecular weight biomolecules such as Polypeptides, proteins, oligonucleotides and synthetic polymers. Can be used along with LC and capillary electrophoresis. Softest ionization technique. Disadvantage: Multiply charged ions are confusing and needs careful interpretation. Sensitive to contaminants such as alkali metals or basic compounds. Not suitable for low polarity compounds.

11. Atmospheric Pressure Chemical Ionization In this method a sample flows through a heated tube where it is volatilized in a mist and sprayed into a corona discharge with the aid of nitrogen nebulization. Corona discharge is used to ionize the analyte in the atmospheric pressure region. APCI is best suited to relatively polar or semi-volatile samples.

12. Atmospheric Pressure Photo Ionization In APPI, samples are ionized by UV light. Molecules with vapors of nebulizer liquid solution, interact with photo beam of UV light. Analyte molecules absorb a photon and become an electronically excited molecule. If the ionization energy of analyte is lower than the energy of photon, then the analyte molecule releases energetic electron and becomes the radical cation and moves forward to the MS analyzer.

13. Matrix Assisted Laser Desorption Ionization Technique In this method ionization is carried out by bombarding a laser beam of sample dissolved in a matrix solution. Matrix is used in MALDI to Absorb the laser energy. Prevent analyte agglomeration. Protect analyte from being destroyed by direct laser beam. Solution of the matrix is made in a mixture of highly purified water and another organic compound (acetonitrile or ethanol). Matrix solution is then mixed with the analyte to be investigated. The organic compound acetonitrile dissolves hydrophobic proteins present in the sample while water dissolves hydrophilic proteins.

14. The solution is then spotted in a air tight chamber on the tip of the sample probe. With a vacuum pump the air is removed and vacuum is created which leads to evaporation of the solvent leaving behind a layer of recrystalized matrix containing analyte molecules. Now the laser beam (EMR) is shooted to the sample, the range of UV radiation used is 360-390nm due to the absorbing substance is present in matrix , it absorbs radiation or energy and thus it transfers some of its energy to sample molecule where by the molecular ions are formed and then accelerate to analyzers.

15. Fast Atom Bombardment It is an ionization technique in which the analyte and non-volatile liquid matrix mixture is bombarded by a high energy beam of inert gas such as Argon or Xenon. This technique is used for ionization of polar high molecular weight compounds such as polypeptides. Commonly used matrices include :- Glycerol Monothioglycerol Carbowax These solvents easily dissolve organic compounds and do not evaporate in vacuum. The bombarding beam consists of Xenon or Argon atoms of high translational energy. This beam is produced by first ionizing the Xenon (or Argon atoms with electrons to give Xenon radical cations. The radical cations are accelerated and are passed through the chamber containing xenon atoms.

16. During this passage high energy cation obtain electrons from Xenon atoms to become high energy atoms (Xe). The lower energy ions are removed by electrostatic deflector. The analyte is dissolved in the liquid matrix such as glycerol and applied as a thin layer on the sample probe shaft. The mixture is bombarded with the high energy beam of Xenon atoms. Xenon ionizes the glycerol molecules to give glycerol ions. These ions react with the surrounding glycerol molecules to produce (G+H) + as reactant ions. The sample molecules then undergo proton transfer or hydride transfer or ion-pair interaction with reactant ions to give quassimolecular or psuedomolecular ions such as (M+H) + , (M-H) - or (M+G+H) + . These ions are then extracted from slit lens system designed to collect ions and directed to mass analyzer.

17. FAST ATOM BOMBARDMENT Advantages: Used for ionization of polar high molecular weight samples. Provides rapid heating of samples and reduces sample fragmentation. Rapid ionization. Disadvantages: Difficult to distinguish between low molecular weight compounds. Compounds must be soluble in liquid matrix. Not good for multiply charged compounds.

18. REFERENCES A. Skoog, F. James Holler and Timothy A. Nieman. Principles of Instrumental analysis, 5 th Edition, 499-511. Willard Meritt . Instrumental Methods Of Analysis, 7 th Edition, 468-474. http://www.chem.ox.ac.uk/spectroscopy/massspec/Lecture/oxmain_lectureCI.html http://www.astbury.leeds.ac.uk (A.E. Ashcroft's MS web pages and tutorial) http://en.wikipedia.org/wiki/Atmospheric_pressure_chemical_ionization

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