Mass Spectrometry

A brief introduction to Mass Spectrometry and its main components.

1. Mass Spectrometry By – Siddharth Kumar Sahu M. Pharmacy

2. Content Introduction Principle Theory Instrumentation Ionisation Technique Applications

3. Mass spectrometry (MS)   is used for determining the masses of particles, to determine the elemental composition of a sample or molecule. . Mass spectrometry is a powerful analytical technique that is used to identify unknown compounds, to quantify known materials, and to elucidate the structure and chemical properties of molecules . Introduction

4. The MS principle consists of ionizing chemical compounds to generate charge molecules or molecule fragments and measurement of their mass-to-charge ratio by using the one of a variety of techniques . A mass spectrometer is a instrument that measures the mass-to-charge (m/z) ratio of ions. (Ionization ) Ionization is a process of charging a molecule. Molecules must be charged in order to measure them using a mass spectrometer Principle

5. The relative abundance of positively charged fragments of various mass-to-charge ratios is the characteristic feature of the molecule that serve to identify the substance. It is determined by = e V [kinetic energy= 1/2 mv2 ] where, m=mass of ion v =velocity of ion V=accelerating voltage e=charge on ion   Theory

6. A mass spectrometer generates multiple ions from the sample under investigation This molecular ion undergoes fragmentation. Each primary product ion derived from the molecular ion, in turn , undergoes fragmentation, and so on The ions are separated in the mass spectrometer according to their mass-to-charge ratio, and are detected in proportion to their abundance. A mass spectrum of the molecule is thus produced. It displays the result in the form of a plot of ion abundance versus mass-to-charge ratio.

7. Instrumentation components are :- Inlet system Ion source / Ionisation chamber Analyser Detector Data system Chemical Ionization Electro Spray Ionization (ESI) MALDI (Matrix Assisted Laser Desorption Ionisation) Atmospheric Pressure Photo Ionization-(APPI) Atmospheric-pressure chemical Ionization (APCI ) Quadrupole Mass Analyzer Orbitrap Analyzer TOF (Time of Flight) Electron Multipliers Faraday Cup Multichannel Plates. Array Detector Vacuum system

8. Schematic diagram -

9. Mass spectrometers can be divided into three fundamental parts, namely the  Ionization Source , the  Analyzer and the  Detector . Ion Source : For producing gaseous ions from the substance being studied. Analyser : For resolving or separates the ions into their characteristics mass components according to their mass-to-charge ratio. Detector System : For detecting the ions and recording the relative abundance of each of the resolved ionic species Instrumentation Fig:- Representative diagram of Mass Spectrometry

10. Chemical ionisation involves the ionisation of a reagent gas, such as methane at relatively high pressure in a simple electron impact source. the reagent gas ions collide with the analyte molecules producing ions through gas phase reaction Reagent gas ionization: CH4 = CH4+ +e– (also CH3+, CH2 +) ESI - The spray is changed into small droplets, and some of the solvent is vaporized using a current of heated gas. The heated droplets are charged and eliminate most of the solvent . Due to ionic repulsion the droplets generate individual ions , most of them still solvated . Multiple charged molecules can also be formed in this process, in particular for compounds such as peptides. Atmospheric Pressure Ionization(API) Source forms gas phase samples ions from sample molecules that are contained in solution. Atmospheric Press. Photoionization technique used to form ions from the analyte molecule. Ion Sources -

11. Schematic diagram of an electrospray ionization (ESI) ESI Source-

12. MALDI (Matrix Assisted Laser Desorption Ionisation) MALDI Technique

13. Mass Analyser - The mass analyser separates the ions formed in the ionization source according to their mass-to-charge (m/z) ratios using some physical property e.g. electric or magnetic fields. Quadrupole Analyser Orbitrap Analyser TOF (Time Of Flight)

14. 1. Quadrupole type mass spectrometers separate the ions by passing them along the central axis of four parallel equidistant rods that have a fixed voltage (DC) and an alternating (RF ) voltage applied to them . The field strengths (voltage) can be set such that only ions of one selected mass can pass through the Quadrupole, while all other ions are deflected to strike the rods.

15. 2. Orbitrap Analyzer The Orbitrap analyzer consists of a small electrostatic device into which ion packets are injected at high energies to orbit around a central, spindle-shaped electrode. The image current of the axial motion of the ions is picked up by the detector and this signal is Fourier transformed (FT) to yield high-resolution mass spectra . It capable of MS/MS using a Quadrupole for precursor-ion selection. Fig. Orbitrap Analyzer

16. 3. TOF Analysers separate ions by time without the use of an electric or magnetic field. In a crude sense, TOF is similar to chromatography, except there is no stationary/ mobile phase, instead the separation is based on the kinetic energy and velocity of the ions. Ions of the same charges have equal kinetic energies; kinetic energy of the ion in the flight tube is equal to the kinetic energy of the ion as it leaves the ion source .

17. A tiny current is produced when the ion reaches the detector. The detector amplifies the signals which are then transmitted to the data system to be represented as peaks on a mass spectrum ( Mass spectrum : graphic representation of ions separated according to their m/z ratio) Some detectors are- Electron Multiplier Faraday Cups Micro channel Plate Detectors Array Detector Mass Detectors

18. Continuous dynode electron multiplier An electron multiplier (continuous dynode electron multiplier ) is a vacuum-tube structure that multiplies incident charges . In a process called secondary emission, a single electron can , when bombarded on secondary emissive material, induce emission of roughly 1 to 3 electrons. If an electric potential is applied between this metal plate and yet another, the emitted electrons will accelerate to the next metal plate and induce secondary emission of still more electrons . This can be repeated a number of times, resulting in a large shower of electrons all collected by a metal anode, all having been triggered by just one 1. Electron Multiplier

19. Faraday cup is part of a circuit where ions are the charge carriers in vacuum and the faraday cup is the interface to the solid metal where electrons act as the charge carriers (as in most circuits ). By measuring the electrical current (the number of electrons flowing through the circuit per second) in the metal part of the circuit the number of charges being carried by the ions in the vacuum part of the circuit can be determined 2. Faraday C up

20. In order to interpret the mass spectrum, one should attain an understanding of the ionisation process . To observe fragmentation pattern . 1.The exact molecular weight : The molecular weight of a pure compound from the identification of the parent peak. The molecular weight one can determine molecular formula. 2.The isotope effects : Heavy isotopes will exhibit peaks in a mass spectrum at m/e one or more units higher than normal. i.e., there will be small peaks at M+1 and M+2 . GENERAL RULES FOR INTERPRETATION OF MASS SPECTRA

21. 3 . The Nitrogen Rule • In organic compounds , is a relationship between the valance and the mass of the most common isotope for most elements . • Even elements have an even valance. • Odd elements have an odd valance. • This leads to the ‘nitrogen rule.’ It assumes that we are limiting our elements to C, H, halogens, O and N . 4 . Molecular formula The molecular formula may often be obtained by high-resolution spectrometer measurements, because atomic weights are not integers. For example, a distinction among CO, N 2 , CH 2 N and C 2 H 4  is possible for nominal weight.

22. Mass spectrometry has both qualitative and quantitative uses . “To identify unknown compound and to Quantify known compound” QUALITATIVE APPLICATIONS Determination of Molecular Weight. Determination of Molecular Formula. Determination of Partial Molecular Formula. Determination of Structure of Compounds. QUANTITATIVE APPLICATIONS Determination of Isotope Abundance. Determination of Isotope Ratio . Differentiation between Cis and Trans Isomers. Determination of Ion-molecule Reactions. Identification of Proteins, Peptides, Polymers. Detection of Impurity. Applications

23. Thermo Fisher Scientific User Guide & Thermo Orbitrap Scigelova . Some Research Papers on mass spectrometry & Metabolism. A Textbook of “Instrumental Method of Chemical Analysis”. B.K. Sharma, Instrumental Methods of Chemical Analysis, Goel Publishing House. Beckett & Stenlake , Practical Pharmaceutical Chemistry, Vol.-II, CBS Publishers & Distribution. www.Slideshare.com Rania Mohamed El— S harkawy , ‘Advances In Medical Research ` From Molecular Medicine-to Clinical Application ` Mass Spectrometric Techniques. K . Rakesh Gupta, Mass Spectrometry. Kommineni , Vidyachowdhary , Basic Principles In Interpretation Of Mass Spectra. Reference’s