Mass spectroscopy

Ionization techniques

1. MASS SPECTROSCOPY 04-11-2019 Mass Spectroscopy 1 PRIYA P.N FIRST SEMESTER MPHARM DEPARTMENT OF PHARMACOLOGY ST JOSEPH COLLEGE OF PHARMACY,CHERTHALA

2. ION SOURCE (IONIZATION CHAMBER) The ions are produced in a box like enclosure called the ion source. The starting point of mass spectroscopic analysis is the formation of gaseous analyte ion. The mass spectra obtained depending on the method used for ionization. Gas phase source Desorption source 04-11-2019 Mass Spectroscopy 2

3. GAS PHASE SOURCE Samples that are volatile or volatilizable outside in source are used. The sample is first vapourised outside the ion source and then ionised. It is limited to thermally stable compound having boiling point less than 500ºc and compound with molecular weight less than 10ˆ3 Dalton. DESORPTION PHASE Solids and liquids are used. Here the sample is vapourised and ionized inside ionization chamber. Applicable to non volatile & unstable compounds and the molecule with a molecular wt. large as 10ˆ5 D. 04-11-2019 Mass Spectroscopy 3

4. Ion source can be also be classified in to two HARD SOURCE SOFT SOURCE HARD SOURCE Impact sufficient energy to the analyte molecule. Mass spectrum with hard source gives information regarding functional groups and thus structural about analyte . SOFT SOURCE Cause little fragmentation, the resulting mass spectrum often consist of the molecular ion peak and only a few any other peak . Soft source give information about the molecular wt. of the analyte molecule. 04-11-2019 Mass Spectroscopy 4

5. GAS PHASE Electro impact ionization Chemical ionization Field ionization DESORPTION PHASE Field desorption Electrospray ionization MALDI Plasma desorption Fast atom bombardment Thermal(surface)ionization 04-11-2019 Mass Spectroscopy 5

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7. It is also know as Electro bombardment or Electro ionization . CONSTRUCTION AND WORKING Electro impact ionization source consist of a ionizing chamber which is maintained at a pressure of 0.005 tore and temperature of 200 ± 0.25 degrees. Electro gun is located perpendicular to chamber. Electrons are emitted from a glowing filament (Tungsten or rhenium) by thermionic emission and accelerated by a potential of 70v applied between the filament and anode. 04-11-2019 Mass Spectroscopy 7

8. These electrons are drawn in the ionization chamber through positively charged slits . The number of electrons is controlled by filament temperature and energy is controlled by filament potential . The sample is brought to a temperature high enough to produce molecular vapours. 04-11-2019 Mass Spectroscopy 8

9. MECHANISM The gaseous sample and the electron coiled at right angles in the chamber and ion are formed by exchange of energy during these collision between electron beam and sample molecule . 04-11-2019 Mass Spectroscopy 9

10. ADVANTAGES Gives molecular mass and also the fragmentation pattern of the sample . Extensive fragmentation and consequent large number of peak give structural information. Give reproducible mass spectra. DISADVANTAGES Sample must be thermally stable & volatile. A small amount of sample is ionized (1in 1000 mole) Unstable molecular ion fragment are formed so readily are absent from mass spectrum. 04-11-2019 Mass Spectroscopy 10

11. CHEMICAL IONIZATION CONSTRUCTION AND WORKING Chemical ionization is carried out in an instrument similar to electron impact ion source with some modification such as; - Addition of a vacuum pump - Narrowing of exist slit to mass analyser to maintain reagent gas pressure of about 1 tore in the ionization chamber. -Providing a gas inlet . 04-11-2019 Mass Spectroscopy 11

12. It is a two part process In the first step * A reagent gas is ionized by electron impact ionization in the source. * The primary ions of reagent gas react with additional gas to stabilized reagent ion. In the second step, the reagent ions interact with sample molecular ions * In this technique the sample is diluted with large excess of reagent gas. Gases commonly used as reagent with low molecular weight compound such as methane, tertiary isobutene, ammonia, nitrous oxide, oxygen, hydrogen. 04-11-2019 Mass Spectroscopy 12

13. TYPES OF CHEMICAL IONISATION Depending upon the ions formed CI is categorised as (1)Positive chemical ionisation (2) Negative chemical ionisation POSITIVE CHEMICAL IONISATION In this technique positive ions of samples are produced. In positive chemical ionisation, gas such as methane, ammonia,isobutane are used. Eg: methane is used as a regent gas. Methane is ionized by electron impact: CH4 +ē CH4+ + 2ē Primary ions react with additional reagent gas molecules to produce stabilized reagent ions: CH4+ + CH4 CH5+ + CH3 CH3+ + CH4 C2H5+ + H2 04-11-2019 Mass Spectroscopy 13

14. NEGATIVE CHEMICAL IONISATION In this technique negative ions of samples are formed. Oxygen and Hydrogen are as reagent gases. The negative ions are formed by following reactions (a) Resonance electron capture M + ē M+ (b) Dissociative electron capture Rcl + ē R + cl- H2O + ē H + OH- 04-11-2019 Mass Spectroscopy 14 The reagent ions then react with the sample molecules to ionize the sample molecules: CH5+ + MH CH4 + MH2+ (Proton transfer) CH3+ + MH CH4 + M+ (Hybride abstraction) CH4+ + MH CH4 + MH+ (Charge transfer)

15. ADVANTAGES Used for high molecular weight compounds . Used for sample which undergo rapid fragmentation in EI . LIMITATION Not suitable for thermally unstable and non volatile sample. Relative less sensitive than EI ionisation. Sample must be diluted with large excess of reagent gas to prevent primary interaction between the electron and sample molecule. 04-11-2019 Mass Spectroscopy 15

16. FIELD IONISATION FI is used produce ions from volatile compounds that don't give molecular ion by EI. It is produce molecular ions with little or no fragmentation . Application of very strong electric field induce emission of electrons. 04-11-2019 Mass Spectroscopy 16

17. CONSTRUCTION & WORKING sample molecule in vapour phase is brought between two closely spaced electrode in the presence of high electric field (10ˆ7-10ˆ8 v/cm). If the metal surface (anode) has proper geometry (a sharp tip, cluster of tips or a thin wire)and is under vacuum (10ˆ-6 torr ) this force is sufficient to remove electrons from the sample molecule without imparting much excess energy. The electric field is produced by applying high voltage (20Kv) to these specially formed emitter (Tungsten). These cations are accelerated out of the source and their mass is analysed by analyser. 04-11-2019 Mass Spectroscopy 17

18. ADVANTAGE As fragmentation is less, abundance of molecular ions (M+) is enhanced, hence this method is useful for relative molecular mass and empirical formula determination . DISADVANTAGES Not suitable for thermally unstable and non volatile sample . Sensitivity is less EI ion source. No structural information is produced as very little fragmentation occurs. 04-11-2019 Mass Spectroscopy 18

19. ELECTROSPRAY IONIZATION Electrospray ionization is a technique used in mass spectrometry to produce ions from macromolecules such as proteins,polypeptiedes and oligonucleotide having molecular wt of 10,000 Da or more. 04-11-2019 Mass Spectroscopy 19

20. CONSTRUCTION AND WORKING The method generate 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/min. The liquid pushes itself out of capillary as a mist or aerosol of fine charged droplets. In the set of aerosol droplets is produced by a process involving formation of taiylor cone and a jet from the tip of the cone. These charged droplets are then passed through desolving capillary where the solvent is evaporated in high vacuum and attachment of charge to the analyte molecule take place. Desolvating capillary uses warm N2 as nebulising gas. 04-11-2019 Mass Spectroscopy 20

21. As the droplets evapourate the analyte molecule comes from closer to together . These molecule become unstable as the similarly charged molecule come closer together and the droplets explode once again, This is called Coulombic fission . These process repeate itself until the analyte is free from solvent and is lone ion . The ion then moves to the mass analyser. ADVANTAGES Most important techniques for analysis of high molecular weight biomolecules such as polypeptide,protein,oligonucleotides and synthetic polymers . Can be used along with LC and capillary electrophoresis . 04-11-2019 Mass Spectroscopy 21

22. 04-11-2019 Mass Spectroscopy 22 FAST ATOM BOMBARDMENT In this 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. Commonly used matrix include: Glycerol,Monothioglycerol,Carbowax,2,4-diphenyl phenol,3-nitrobenzyl alcohol These solvent easily dissolve organic compound and don't evapourate in vacuum.

23. 04-11-2019 Mass Spectroscopy 23 CONSTRUCTION &WORKING The bombarding beam consist of xenon or argon atoms of high translational energy. The beam is produced by first ionizing the xenon or Argon atom with electron to give xenon radical cation. Xe + ē Xe + 2ē The radical cations are then accelerated to 6-10 Kev to give radical cations of high translational energy (Xe)++,which are then passed through a chamber containing Xenon atoms at a pressure of 10ˆ-5 tore. During this passage high energy cation obtain electron from Xenon atom to become high energy atom (Xe). The lower energy ions are removed by electrostatic deflector. (Xe)++ Xe+ +Xe (Xe)+ + Xe (Xe) + Xe+

24. 04-11-2019 Mass Spectroscopy 24 MATRIX PREPARATION 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 atom . Xenon ionize the glycerol molecule to give glycerol ions. These ions react with the surrounding glycerol molecules to produce( G+H)+ as recent ions. The sample molecule then undergo proton transfer or hybride transfer or ion-pair interaction with reactant ions so give quassimolecular or pseudo molecular ion such as (M-H),H+,(M+). These ions are then extracted from slit lens system designed to collect ions and directed to mass analyser.

25. 04-11-2019 Mass Spectroscopy 25 ADVANTAGES Used for ionization of polar high molecular wt sample . Provide rapid heating of sample and reduces sample fragmentation. Rapid ionization. DISADVANTAGES Difficult to distinguish b/w low molecular wt compounds. Compound must be soluble in liquid matrix. Not good for multiply charged compounds.

26. 04-11-2019 Mass Spectroscopy 26 ATMOSPHERIC PRESSURE CHEMICAL IONIZATION APCI produce ions using a reagent gas generated from solvent vapour. CONSTRUCTION &WORKING The solvent a mixture of methanol,acetonitrile,water at 0.5ml/min is supplied to the probe by a pump. Liquid spray is produced by passing co-axial nebuliser gas (N2). The solvent spray is vapourised by a heating. Once formed, the vapour is emanating from a corona pin held at 3 Kv .

27. 04-11-2019 Mass Spectroscopy 27 Ion/molecule reaction then result in the formation of a reactive species. The sample ions are then accelerated out of the atmospheric pressure source and in to the mass analyser by application of small voltage (typically 20-70 V) to the skimmer cone. The pressure differential between source and analyser region is maintained by the pressure of an area of intermediate vacuum. During the ionization process itself, little energy is transferred to the sample molecule, and fragmentation is minimal. APPLICATIONS APCI is suitable for the analysis of organic compounds with medium- high polarity. Since positive ionization is dependent on protonation, molecule containing basic functional groups such as amino, amide ester, aldehyde/ketone and hydroxyl can be analysed.

28. 04-11-2019 Mass Spectroscopy 28 MATRIX ASSISTED LASER DESORPION (MALDI) Is a technique in mass spectrometry for ionization of biomolecules (polymer such as protein, polypeptide and sugar)and synthetic polymers that are more fragile and form fragments when ionized, by convential methods. (a)MATRIX --Matrix is used in MALDI to *Absorb the laser energy . * Prevent analyte agglomeration . *Protect analyte from being destroyed by direct laser beam. --Matrix consist of a crystallised molecules of which the most commonly based are; *3,5 dimethoxy-4-hydroxyl cinnamic acid * α -cyano-4-cinnamic acid compound *2,5-dihydroxy benzoic acid

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30. 04-11-2019 Mass Spectroscopy 30 Preparation of matrix : (a)Solution of the matrix is made is made in mixture of highly purified water and another organic compound. (b)Trifluroacetic acid is also added . (c) Solution is the spotted in a air tight chamber on the tip of sample probe . (d)When a vacuum pump the air is removed and vacuum is created which leads to evaporation of the solvent leaving behind a layer of recrystallized matrix containing analyte molecules. ( b)LASER The solid mixture is exposed to pulsed laser beam. The matrix absorbs the laser energy and transfer some of this energy to the analyte molecules which results in the sublimation of sample molecules as ions or the matrix after absorbing the laser gets ionized and transfer part of this charge to the sample molecule &ionize it.

31. 04-11-2019 Mass Spectroscopy 31 Nitrogen or carbon lasers are most commonly used. The ions produced in this process are quassimolecular ions that are ionized by addition of proton (M+H)+ . It generally produces singly charged ions in some cases doubly charged ions such as (M+2H)2+ are also observed. The chamber consist of two electrode and the ions are produced b/w the electrode. When the polymers from cations the cathode is placed right behind the sample and anode in front of the sample. The cations get attached towards the negatively charged .

32. 04-11-2019 Mass Spectroscopy 32 USES OF MALDI Used to characterize and identify large molecules. Used in pharmaceutical for QC,monitoring of enzyme reactions. Used in DNA sequencing for forensic . Used to identify different strain of viruses to help develop vaccines.

33. 04-11-2019 Mass Spectroscopy 33 1.Spectrometric identification of organic compounds – Silverstein and Webster. 2.Organoc spectroscopy – William Kemp. 3.Organic Spectroscopy – B K Sharma. 4.Mass spectroscopy – Davis Martin and Frearon. 5.Internet sources REFERENCES

34. THANK YOU ! 04-11-2019 Mass Spectroscopy 34