Mass Spectrometry by ANITHA SRI

OVERVIEW OF MASS SPECTROMETRY : 

OVERVIEW OF MASS SPECTROMETRY M. Anitha Sri (Y11MPH448) I/II M.Pharmacy, Industrial pharmacy CHALAPATHI INSTITUTE OF PHARMACEUTICAL SCIENCES.

Contents : 

Contents Introduction Instrumentation Mass Spectrum Resolution Determination of molecular formula Data analysis and interpretation Applications 2

INTRODUCTION : 

INTRODUCTION A mass spectrometer is an instrument that measures the masses of individual molecules. Three Basic functions: 1. creating gaseous ion fragments from the samples 2. separating them according to their mass-to-charge ratio 3. records the relative abundance of each ionic species present Also known as positive ion spectra or line spectra. 3

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Block diagram of Components of Mass Spectrometer

INSTRUMENTATION : 

INSTRUMENTATION Inlet system Ion source Electrostatic accelerating system Magnetic field Ion separator Ion collector and Detector Vacuum system 5

Inlet system : 

Inlet system Direct vapor inlet Direct insertion probe Gas chromatography(GC-MS) Liquid chromatography(LC-MS) Particle Beam Interface Thermospray Interface Electrospray Interface Desorption techniques(FAB and LSIMS) 6

Direct Vapour Inlet : 

Direct Vapour Inlet Gases or volatile liquids Method is Molecular leak or Molecular pumping The sample can be introduced through a septum port or through a valve port. 7

Direct Insertion Probe : 

Direct Insertion Probe 8 Solids and liquid samples. Autoprobe.

GC-MS : 

GC-MS Most common technique for introducing samples. Several different interface designs are used to connect these two instruments. The MS coupled to the GC should be capable of high resolution. Highly specific. 9

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10

LC-MS : 

LC-MS Used for Thermo labile compounds. Several interfaces are used to connect LC and MS. 11

Particle Beam Interface : 

Particle Beam Interface Solvent is removed from an aerosol of LC effluent The resulting analyte is analysed in the ion source Known as MAGIC (Monodisperse Aerosol Generator Interface for Chromatography) 12

Thermospray : 

Thermospray Involves simply heating the tip of the entry tube to promote vaporisation. Through the centre of the stainless steel tube, passes a small diameter tube which carries the column eluent. The tube projects slightly beyond the end of the heater cap which is situated in a cartridge heater together with a thermocouple. 13

Electro Spray : 

Electro Spray 14 Sample is dissolved in a solvent and pumped through a narrow capillary. Voltage is applied to the capillary tip and the sample is dispersed into an aerosol, aided by a coaxially introduced nebulising gas.

ESI : 

ESI 15 The charged droplets diminish in size by solvent evaporation assisted by a flow of drying gas. Eventually charged sample ions, free from solvent, are released from the droplets, which pass through the orifice into an intermediate vacuum region and from these through a small aperture into the analyser of the MS.

Ionisation Techniques : 

Ionisation Techniques 16

Electron Impact : 

Electron Impact 17

Chemical Ionisation : 

Chemical Ionisation Chemical interaction between reagent gas ions and analyte molecule. Two-step process. CH4 + e- = CH4+ + 2e- Secondary ions of reagent gas are produced, which react with the analyte molecules. The mechanism may be proton transfer, hydride abstraction or charge transfer. CH4+ + MH = CH4 + MH+ CH5+ + MH = CH4 + MH2+ CH3+ + MH = CH4 + M+ Reagent gases: Argon, Helium, Nitrogen. 18

Field Ionisation : 

Field Ionisation 19 Ions are formed under the influence of high electric field produced by applying high voltages. On the surface of fine tube, many hundreds of projecting carbon microtips are present. These extract the electron from the sample and ionise the sample molecules.

Fast Atom Bombardment : 

Fast Atom Bombardment 20 High energy primary beam is directed at a target surface to obtain high yield of secondary ions. Primary beam may be ions, electrons, photons or neutral atoms. SIMS may be dynamic or static.

MALDI : 

MALDI Two step process. Desorption is triggered by a laser beam. The second step is ionization.  Nitrogen laser of 337 nm wavelength is used. Sinapinic acid is used as matrix for proteins and α-cyano-4-hydroxycinnamic acid for peptides. 21

Choosing an Ionisation technique : 

Choosing an Ionisation technique 22

Electrostatic Accelerating system : 

Electrostatic Accelerating system 23 The positive ions formed in the ionisation chamber are accelerated by pairs of accelerator plates to impart velocities to the ions. Ions are sorted acc. to m/e ratio based on 3 properties: energy, velocity and momentum. The beam from the slits of these plates consists of a collimated ribbon of ions having equal energies. K.E = eV = ½ m1v12 = ½ m2v22……….

Magnetic Field : 

Magnetic Field eV = ½ mv2 F = HeV HeV = mv2/r m/e = H2r2/2V 24 e = charge m= mass v = velocity V = voltage F = Magnetic force H = Magnetic field strength r = radius

Ion Separator : 

Ion Separator Single Focussing Double Focussing Cycloidal Quadrupole TOF MS/MS Radio Frequency 25

Single Focussing : 

Single Focussing 26

Single Focussing : 

Single Focussing 27

Double Focussing : 

Double Focussing 28

Cycloidal : 

Cycloidal 29

Quadrupole : 

Quadrupole 30

Time of Flight : 

Time of Flight The time-of-flight (TOF) mass analyzer separates ions in time as they travel down a flight/drift tube. This is a very simple mass spectrometer that uses fixed voltages and does not require a magnetic field. The greatest drawback is that TOF instruments have poor mass resolution, usually less than 500. 31

MS/MS : 

MS/MS 32 Hybrid MS. The two analysers are separated by a field free collision chamber, which contains an inert gas.

Radio Frequency : 

Radio Frequency 33 An assembly of grids is employed to select ions acc. to their velocities. Alternative grids are connected to a radiofrequency source and the other grids are connected to a steady potential. It is simple in construction and doesn’t require a magnet.

Ion collector and Detector : 

Ion collector and Detector Detection of ions is based on their charge Detectors monitors the ion current, amplifies it and the signal is transmitted to the data system where it is recorded in the form of mass spectra. Types of Detectors: Faraday Cup Collector. Electron Multiplier Channel Electron Multiplier Array The detection is either by pulse counting or analog measurement. 34

Faraday-Cup : 

Faraday-Cup 35 Ions enter the cup and transfer their charge to the cup. Secondary electrons are generated. No. of secondary electrons generated depends on several factors: mass of ions energy of ions charge on the ions Angle of incidence material of cup nature of the ion.

Electron Multiplier : 

Electron Multiplier 36 A metal plate called conversion dynode that converts the impinging ions to electrons is present. Ion beams strikes the conversion dynode. Secondary electrons are produced by the electron multiplier.

Channel Electron Multiplier Array : 

Channel Electron Multiplier Array 37 Composed of a regular close packed array of channels in a flat plate of semiconducting material. Inside of each channel is coated with a secondary electron emissive material, thus each channel constitutes an independent electron multiplier.

Vacuum system : 

Vacuum system All mass spectrometers operate at very low pressure (high vacuum). This reduces the chance of ions colliding with other molecules in the mass analyzer. Any collision can cause the ions to react, neutralize, scatter, or fragment. All these processes will interfere with the mass spectrum. To minimize collisions, experiments are conducted under high vacuum conditions, typically 10-2 to 10-5 Pa (10-4 to 10-7 torr) depending upon the geometry of the instrument. This high vacuum requires two pumping stages. The first stage is a mechanical pump that provides rough vacuum down to 0.1 Pa (10-3 torr). The second stage uses diffusion pumps or turbo molecular pumps to provide high vacuum. 38

General Fragmentation Patterns : 

General Fragmentation Patterns Simple Direct cleavage Retro-Diels Alder Reaction Hydrogen Transfer Rearrangement Mc lafferty rearrangement 39

Mc-Lafferty rearrangement : 

Mc-Lafferty rearrangement 40 Involves intramolecular migration of γ-hydrogen from electron rich center to electron deficit center followed by cleavage at β position resulting in the formation of neutral alkene. Common in ketones, esters and carboxylic acids

Types of ions : 

Types of ions Molecular ion Fragment ions Rearrangement ions Multiply charged ions Negative ions Metastable ions Pseudomolecular or Quasi ions 41

Metastable ions : 

Metastable ions Ions formed in the analyser after moving away from the ionisation chamber. Gives broad bands. Formed at non-integral mass numbers. Mass of metastable ion is calculated by: m* = m22/m1 m* = mass of metastable ion m1 = mass of molecular ion m2 = mass of daughter ion 42

Derivitisation : 

Derivitisation For some ionisation techniques, the compound should be derivitised before being analysed. Derivitisation is the use of chemicals to modify the analyte, usually to reduce its polarity. Often OH and NH groups are reacted with silylating reagents, or acetic anhydride, to form compounds with O-Si, N-Si, O-C or N-C bonds instead. The derivative then lacks the ability to form hydrogen bonds and is more volatile than the analyte was. Mass spectrometry is a gas phase technique; irrespective of the nature of the sample the analysis is on gaseous ions, hence the need for volatility. 43

Mass Spectrum : 

Mass Spectrum The mass spectrum is presented in terms of ion abundance vs. m/e ratio (mass) The most abundant ion formed in ionization gives rise to the tallest peak on the mass spectrum – this is the base peak 44 base peak, m/e 43

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All other peak intensities are relative to the base peak as a percentage If a molecule loses only one electron in the ionization process, a molecular ion is observed that gives its molecular weight – this is designated as M+ on the spectrum 45 M+, m/e 114

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In most cases, when a molecule loses a valence electron, bonds are broken, or the ion formed quickly fragment to lower energy ions. The masses of charged ions are recorded as fragment ions by the spectrometer – neutral fragments are not recorded ! 46 fragment ions

Resolution : 

Resolution 47 Adjacent peaks must be clearly separated. The valley between the two adjacent peaks should not be more than 10% of the height of the larger peak. R = Mn/Mn - Mm

Determination of Molecular Formula : 

Determination of Molecular Formula Nitrogen Rule Rule Of Thirteen When a molecular mass, M+, is known, a base formula can be generated from the following equation: M/13 = n + r/13 the base formula being: CnHn+r Index of Hydrogen Deficiency: HDI = n-r+2 / 2 Ring rule: For the molecule CwHxNyOz, R = w + 1 + y-x/2 48

Data Analysis from mass spectrum : 

Data Analysis from mass spectrum The molecular ion peak in aromatic compounds is relatively much intense. Conjugated olefins show more intense molecular ion peak as compared to the corresponding non-conjugated olefins with same no. of unsaturation. The relative abundance of the saturated hydrocarbon is more than the corresponding branched chain compound. In aromatic compounds, the substituent groups like -OH, -OR, -NH2 increase the relative abundance and –NO2, -CN decrease the relative abundance. 49

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Absence of molecular ion peak in the mass spectrum means that the compound under examination is highly branched or tertiary alcohol. In case of Chloro or Bromo compounds, isotope peaks(M+ + 2) are also formed along with the molecular ion peak. Isotope peak is not observed when Fluorine or Iodine atom is present in the compound. 50

Computerised matching of spectra with spectral libraries : 

Computerised matching of spectra with spectral libraries The computer can compare a mass spectrum it has determined with the spectra in the databases of the libraries. The output is a table called “HIT LIST”. Hit list includes the name of each compound that the computer has used for matching, its molecular weight, molecular formula, probability that the spectrum of the test compound matches the spectrum in the data base. The probability is determined by the no. of peaks and their intensities that can be matched. 51

Applications : 

Applications Determination of molecular mass and structure. Determination of Isotopic abundance. Distinction between isomers. Determination of Ionisation potential and Bond Dissociation energies. Detection of presence of impurities. Identification of unknown compound. 52

References : 

References U.S.P. Y.R. SHARMA. ELEMENTARY ORGANIC SPECTROSCOPY, PRINCIPLES AND CHEMICAL APPLICATIONS. 4th ed. S.CHAND. 2007. D.A.SKOOG, F.J. HOLLER, S.R.CROUCH. PRINCIPLES OF INSTRUMENTAL ANALYSIS. 6th ed. THOMPSON BROOKS. 2007. D.L.PAVIA, G.M.LAMPMAN, G.S.KRIZ. INTRODUCTION TO SPECTROSCOPY. 3rd ed. THOMPSON BROOKS. 2001. G.R.CHATWAL, S.K.ANAND. INSTRUMENTAL METHODS OF CHEMICAL ANALYSIS. 5th ed. HIMALAYA PUBLISHING HOUSE. 2002. H.HWILLARD, L.L.MERRITT, J.A.DEAN, F.A.SETTLE. INSTRUMENTAL METHODS OF ANALYSIS. 7th ed. CBS PUBLISHERS.

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