Ionization techniques in lcms.ppt

Brief introduction to LC-MS & ionization techniques used LC-MS

1. Ionization Techniques in LC-MS A Seminar as a part of curricular requirement for I year M. Pharmacy I semester Presented by Mr. G Chiranjeevi (Reg. No. 20L81S0706) Pharmaceutical analysis Under the guidance/Mentorship of Dr. P. Ramalingam, Ph.D Director - R&D Cell , Professor of pharmaceutical analysis And medicinal chemistry

2. 2 Introduction to LC-MS Problems in combining HPLC & MS Interface in LC-MS Types of ionization techniques Choice of ionization technique References contents

3. 3 It is an analytical chemistry technique. combination of physical separation capability of LC (or HPLC) and mass analysis capability of MS. It is a method that combines the separation power of HPLC with detection power of mass spectroscopy. In LC-MS we remove the detector from the column of LC and fit the column to interface of MS. In the most of the cases the interface used in LC-MS are ionization source. Introduction to LC-MS

4. 4 HPLC is a method for separating a complex mixture in to its components. High sensitivity of mass spectroscopy provides the information for identification of compounds or structural elucidation of compounds. As the metabolites appear from the end of the column they enter the mass detector . Where the solvent is removed and the metabolites are ionized.

5. 5 MS work by ionizing molecules and then sorting and identifying the ions according to their mass-to-charge ratio(m/z).

6. 6 Problems in combining HPLC & MS HPLC MS Liquid phase operation 25 – 50 deg Celsius No mass range limitations Inorganic buffers 1 ml/min eluent flow is equivalent to 500ml/min of gas Vacuum operation 200 – 300 deg Celsius Up to 4000 Da for quadrupole mass spectroscopy Require s volatile buffers Accepts 10 ml/min gas flow

7. 7 LC-MS system include a device for introducing samples an interface for connecting such device. If the LC unit is simply connected directly to the MS unit , the liquid mobile phase would vaporize, resulting in large amount of gas being introduced into the MS unit. This would decrease the vacuum level and prevent the target ions from reaching the detector. So interfaces to be used. In this interface neutral liquid molecules converted to gas phase ions also. Interface in LC-MS

8. 8 Mass spectrum is significantly depends up on the ionization method Variations in the spectrum is introduced in the form of 1. number of peaks 2. intensity of peaks It can be can be categorized into two parts Hard ionization technique - high energy & increased fragmentation Soft ionization technique - low energy & decreased fragmentation Types of ionization techniques

9. 9 Thermospray ESI APCI APPI Electron ionization(EI) Chemical ionization(CI) Field desorption FAB MALDI

10. 10 It is also known as electron bombardment ionization. Main function of this technique is to convert the gaseous sample into molecular ions. It is hard ionization technique because it will produce 70eV. Ionization potential of organic compounds are approximately 8eV - 15eV. Due to high energy, large number of fragment ions produced from molecule ion. Electron ionization(EI)

11. 11 Operating at 10-6 tarr Electron ionization process Electron ionization process

12. 12 It is a gaseous phase method and soft ionization technique. Less fragmentation & give intensive peak of molecular ion( i.e more no. of M + ) Some molecules like alcohols, ethers, esters, amino acids etc.. Are highly fragmented in EI. So molecular peaks will not be detected To get proper molecular peaks we are using CI Chemical ionization (CI)

13. 13 Steps of chemical ionization 1) Introduction of carrier/reagent gas into the ionization source at slightly high pressure (1 torr) Ex. Methane, ammonia, isobutane 2) Ionization of carrier gas by electron impact from the ionization source CH 4 + e - CH 4 . + + 2e - CH 4 . + CH 3 + + H + 3) CH 4 . + & CH 3 + are primary ions, these will react with excess CH 4 & it will produce different type of secondary ions.(i.e. CH 5 + , C 2 H 5 + , C 3 H 5 + )

14. 14 CH 3 . CH 4 . + + CH 4 CH 5 + + CH 5 + + CH 4 C 2 H 5 + + H 2 C 2 H 5 + + CH 4 C 3 H 5 + + 2H 2 4) Secondary ions will react with analyte molecule & form ions by three ways. a)Proton transfer b)Hydride transfer c) Electrophillic addition M + CH 5 + [M-H] + + CH 4 M + C 2 H 5 + [M-H] + + C 2 H 4 MH + C 2 H 5 + [M-1] - + C 2 H 6 M + CH 3 + [M-CH 3 ] + or [M+15] +

15. 15 It is a soft ionization technique under desorption method. FAB can be used up to 10,000 Da molecular weight. It is generally used for the determination of mol.wt of peptides. Fast atom bombardment (FAB)

16. 16 Characteristics of the matrix It should be non volatile Less vapour pressure liquids Ex. Glycerol, Thioglycerol, 3-Nitrobengyl alcohol, Diethanolamine & Triethanolamine.

17. 17 It is a soft ionization technique under desorption ionization method. which uses pulsed LASER (Light Amplification by Stimulated Emission of Radiation) beam. It used to determine the mol.wt of peptides, antibodies, protein, molecules etc. Up to the size of 300Da. Matrix assisted LASER desorption ionization (MALDI) - - - - - - - - - - - - - - - vv vv vv - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - vv vv v v vv + Sample sol n 0.1 mg/ml Matrix sol n 10 mg/ml Mixture sol n Co-crystallization Crystallized sample-matrix mixture Ratio 1 : 1000 to 10000 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

18. 18 Matrix materials :- Nicotinic acid, DHB, cinnamic acid derivatives. Matrix material should have low mol.wt, acidic nature, strong laser beam absorption polar functional groups. 0.5 – 20 nano sec. Exposure time by Protonation (ToF)

19. 19 LASER beams :- 337nm:-Nitrogen laser of UV-range 355nm:- frequency tripled Nd : YAG (Neodymium : Yherium, Aluminium, Garnet) 326nm:- frequency quadrupled Nd : YAG 294nm:- IR laser produced by Er : YAG

20. 20 Electrospray ionization(ESI)

21. 21 It is softest ionization method , used for highly polar, least volatile/thermally unstable compounds. Ex. peptides, proteins, lipids, oligosaccharides.

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24. 24 It is based on the mechanism of evaporation and carried out at atmospheric pressure. Combination of CI & ESI. Generally linked with chromatographic instrument like HPLC. Sample injected throw capillary tube, converted into sprayed droplet & finally analyte. Solvent vapour due to heating by N 2 . We use corona discharge needle or β -particle emitter for ionization, it will ionize the solvent vapour molecule. Some times analyte matter also may ionized by the electrode. Atmospheric pressure chemical ionization (APCI)

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26. 26 Due to collision of ion molecule charge transfer b/w solvent and analyte takes place & analyte ion produced. APCI may produce both +ve [MH] + & -ve [M-1] - ions. It used to analyse polar, thermostable substance with molecular weight less than 1500Da.

27. 27 It is based on the mechanism of evaporation and carried out at atmospheric pressure. APPI is similar to APCI, but ionization in APPI is due to photons generated by UV light krypton lamp. Sample injected throw capillary tube, converted into sprayed droplet & finally analyte. Solvent vapour due to heating by N 2 . Desolvation gas (heated N 2 ) will be supplies which will convert the sprayed droplets into the form of vapours of analyte and solvent. Atmospheric pressure photoionization (APPI)

28. 28 Photons emitted by the krypton lamp have specific energy i.e. 10eV Which is sufficient to ionize other atmospheric gas present, due to low energy. Photons of 10eV will not ionize other atmospheric gas present, due to low energy.

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30. 30 Photons will ionize the analyte by three mechanisms 1)Direct APPI:- 2)Indirect APPI:- 3)Dopant assisted APPI :- Toluene is used as dopant to increase the percentage of molecular ion. M + hv M . + + e - S + hv S . + + e - M + S . + M + + S . D + hv D . + + e - M + D . + M + + D .

31. 31 ESI - polar compounds - Large molecules, proteins, polymers - Fragile molecules APCI - Low polarity - < 2000u - Small molecules APPI/ASAP - Non polar molecules - ASAP can analyse low polar high mass molecule (pyrolysis) Choice of ionization technique Atmospheric solid analyse probe (ASAP)

32. 32 Thank you