Mass spectrometry

MASS SPECTROMETRY , FAB, MALDI,PRICIPLE, TYPES OF IONIZATION , APLICATIONS

1. MASS SPECTROMETRY Presented by: Shubham Sharma Pharmaceutical Chemistry Roll no. : 20029 FAB AND MALDI Presented to : Dr. Poonam Piplani Professor ,UIPS, Panjab University

2. CONTENTS Definition Principle Instrumentation Components of MS Ion Source & Types of ionization Ionization Methods Fast Atom Bombardment (FAB) Matrix Assisted Laser Desorption /Ionization (MALDI) MASS ANALYZERS APPLICATIONS OF MASS SPECTROMETRY

3. INTRODUCTION Mass Spectrometry (MS) is a technique for the analysis of a substance in which the molecule is subjected to bombardment by high-energy electrons or atoms to cause ionization and fragmentation to give a series of ions in the gas phase that constitutes the fragmentation pattern observed by using a mass spectrometer . It is a process, which used to identify chemicals in a substance by their mass and charge . Mass spectrometers are instruments that measure mass and charge of molecules. A mass spectrometer also can determine how much of a compound is present in a mixture, also known as mass spectroscopy.

4. PRINCIPLE OF MASS SPECTROMETRY 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.

5. INSTRUMENTATION The instrument used in MS is called M ass spectrometer . It produces a mass spectrum that plots the mass-to- charge (m/z) ratio of compounds in a mixture. Mass spectrometry is used in many different fields and is applied to pure samples as well as complex mixtures . Mass Spectrometry (MS) is used for both qualitative and quantitative chemical analysis .It may be used to identify the elements and isotopes of sample, to determine the masses of molecules, and as a tool to help identify chemical structures. It can measure sample purity and molar mass.

6. COMPONENTS OF MS

7. ION SOURCE AND IONIZATION TYPES Molecular ions are formed when energy of the electron beam reaches to 10-15 eV. Fragmentation of the ion reaches only at higher bombardment energies at 70 eV. IONIZATION TYPES GAS PHASE IONIZATION DESORPTION TECHNIQUE GASES AND VAPOUR LIQUID AND SOLID

8. GAS PHASE IONIZATION Samples are ionized outside the ion source. 1.Electron impact ionization [EIS] 2.Chemical ionization [CI] 3.Field ionization [FI] DESORPTION TECHNIQUE Samples are ionized outside the ion source. 1.Fast atom bombardment [FAB] 2.Matrix assisted desorption [MALDI] 3.Field desorption [FD]

9. IONIZING AGENT

10. IONIZATION METHOD

11. MATRIX ASSISTED LASER DESORPTION [MALDI] In this method ionization is carried out by bombarding a laser beam on the sample dissolved in a matrix solution. It is also known as Soft ionization technique.

12. Matrix is used in MALDI to Absorb the laser energy. Prevent analyte agglomeration. Protect analyte from being destroyed by direct laser beam. MATRIX Matrix consists of a crystallized molecules of which the most commonly used are :- 3,5 – dimethoxy – 4 – hydroxy cinnamic acid (sinapinic acid) α – cyano – 4 – cinnamic acid ( α – cyano or α – matrix). 2,5 – dihydroxy benzoic acid (DHB) .

13. They are of a fairly low molecular weight (to allow easy vaporization), but are large enough (with a low enough vapor pressure) not to evaporate during sample preparation or while standing in the spectrometer. They are often acidic, therefore act as a proton source to encourage ionization of the analyte. They have a strong optical absorption in either the UV or IR range, so that they rapidly and efficiently absorb the laser irradiation. They are functionalized with polar groups, allowing their use in aqueous solutions . They typically contain a chromophore. CONSIDERATIONS

14. Solution of the matrix is made in a mixture of highly purified water and another organic compound (acetonitrile or ethanol). Triofluoro acetic acid (TFA) is also added. If sinapinic acid is used as a matrix the solution is prepared by adding: 20 mg/ml of sinapinic acid. Water : acetonitrile: TFA (50:50:0.1). 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 . This solution is spotted into a MALDI plate. With a vacuum pump the air is removed and vacuum is created which leads to evaporation of the solvent The solvents vaporized , leaving only re- crystallised matrix, but now with analyte molecules embedded into MALDI crystals. The matrix and analyte are said to be co- crystallised. Co-crystallization is a key issue . Continued…

15. Laser: MALDI techniques typically employ the use of UV lasers such as nitrogen lasers (337 nm) and frequency tripled and quadrupled Nd:YAG lasers (355 nm and 266 nm respectively). Although not as common, infrared lasers are used due to their softer mode of ionization . IR-MALDI also has the advantage of greater material removal (useful for biological samples), less low- mass interferences, and compatibility with other matrix-free laser desorption. 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 ablation of the upper layer of the matrix material. Hot plume gets produced during ablation. Second, the analyte molecules are ionized in the hot plume. Ablated species may participate in the ionization where by the molecular ions are formed and then accelerate to analyzers.

16. The mechanism of MALDI Done in three steps.. Formation of a Solid Solution ( ii) Matrix excitation (iii) A nalyte ionization

17. It is essential for the matrix to be in access thus leading to the analyte molecules being completely isolated from each other. This eases the formation of the homogenous 'solid solution' required to produce a stable desorptionof the analyte. Formation of a Solid Solution The laser beam is focused onto the surface of the matrix-analyte solid solution . The chromophore of the matrix couples with the laser frequency causing rapid vibrational excitation, bringing about localized disintegration of the solid solution. The clusters ejected from the surface consists of analyte molecules surrounded by matrix and salt ions. The matrix molecules evaporate away from the clusters to leave the analyte in the gas phase. 2. Matrix Excitation

18. The photo-excited matrix molecules are stabilised through proton transfer to the analyte. Cation attachment to the analyte is also encouraged during this process. yet is also encouraged during this process. It is in this way that the characteristic [M+X ], (X= H, Na, K etc.) analyte ions are formed. These ionization reactions take place in the desorbed matrix-analyte cloud just above the surface. Analyte Ionisation Uses of MALDI :- Used to characterize and identify large molecules Used in pharmaceutical for QC, Monitoring of enzyme reactions Used in DNA sequencing for forensics Used to identify different strains of viruses to help develop vaccines.

19. Sample target for a MALDI mass spectrometer The type of a mass spectrometer most widely used with MALDI is the TOF (time-of-flight mass spectrometer), mainly due to its large mass range. The TOF measurement procedure is also ideally suited to the MALDI ionization process since the pulsed laser takes individual 'shots' rather than working in continuous operation. MALDI-TOF instruments are typically equipped with an "ion mirror", deflecting ions with an electric field, thereby doubling the ion flight path and increasing the resolution. Today commercial reflectron TOF instruments reach a resolving power m/Δm of well above 20,000 FWHM ( full-width half- maximum , Δm is defined as the peak width at 50% of peak height. ) MALDI-FT-ICR MS has been demonstrated to be a useful technique where high resolution MALDI-MS measurements are desired. MASS SPECTROMETER

20. The sample preparation for MALDI is important for both sensitivity, reproducibility and quantification of mass analysis. Inorganic salts which are also part of protein extracts interfere with the ionization process. The salts can be removed by solid phase extraction or by washing the dried droplet MALDI spots with cold water. Both methods can also remove other substances from the sample. Reproducibility and performance What’s its future? Will help revolutionize the medical world and will help lead to treatments for many diseases . Will be useful for DNA sequencing, thus can be useful for forensic investigations.

21. 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. Fast Atom Bombardment

22. This technique is used for ionization of polar high molecular weight compounds such as polypeptides. Commonly used matrices include :- Glycerol Monothioglycerol Carbowax 2,4 – dipentyl phenol 3 – nitrobenzyl alcohol (3 – NBA) 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 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 10-5 torr. Xe + e- = Xe.+ +2e- Continued..

23. 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.

24. Used for ionization of polar high molecular weight samples. Provides rapid heating of samples and reduces sample fragmentation . Rapid ionization. Advantages D isadvantages Difficult to distinguish between low molecular weight compounds. Compounds must be soluble in liquid matrix. Not good for multiple charged compounds .

25. REFRENCES Silverstein, r.M ., Webster, francis X., Kiemle , david J., Spectroscopic identification of organic compounds. John wiley and sons , inc. Chatwal r gurdeep , anand k.Sham,instrumental method of chemical analysis, himalaya publishing house,2.274.

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