![RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14
CH3
.
CH4
.+ + CH4 CH5
+ +
CH5
+ + CH4 C2H5
+ + H2
C2H5
+ + CH4 C3H5
+ + 2H2
4) Secondary ions will react with analyte molecule & form ions by
three ways.
a)Proton transfer
b)Hydride transfer
c)Electrophillic addition
M + CH5
+ [M-H]+ + CH4
M + C2H5
+ [M-H]+ + C2H4
MH + C2H5
+ [M-1]- + C2H6
M + CH3
+ [M-CH3]+ or [M+15]+](https://image.slidesharecdn.com/ionizationtechniquesinlcms-210518144352/85/Ionization-techniques-in-lcms-ppt-14-320.jpg)
![RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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.](https://image.slidesharecdn.com/ionizationtechniquesinlcms-210518144352/85/Ionization-techniques-in-lcms-ppt-26-320.jpg)
Ionization techniques in lcms.ppt
- 1. Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5 MS work by ionizing molecules and then sorting and identifying the ions according to their mass-to-charge ratio(m/z).
- 6. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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 • Requires volatile buffers • Accepts 10 ml/min gas flow
- 7. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9 methods Gas phase Desorption Evaporation Thermospray ESI APCI APPI Electron ionization(EI) Chemical ionization(CI) Field desorption FAB MALDI
- 10. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11 Operating at 10-6 tarr Electron ionization process Electron ionization process
- 12. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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 CH4 + e- CH4 .+ + 2e- CH4 .+ CH3 + + H+ 3) CH4 . + & CH3 + are primary ions, these will react with excess CH4 & it will produce different type of secondary ions.(i.e. CH5 +, C2H5 +, C3H5 +)
- 14. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14 CH3 . CH4 .+ + CH4 CH5 + + CH5 + + CH4 C2H5 + + H2 C2H5 + + CH4 C3H5 + + 2H2 4) Secondary ions will react with analyte molecule & form ions by three ways. a)Proton transfer b)Hydride transfer c)Electrophillic addition M + CH5 + [M-H]+ + CH4 M + C2H5 + [M-H]+ + C2H4 MH + C2H5 + [M-1]- + C2H6 M + CH3 + [M-CH3]+ or [M+15]+
- 15. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 16 Characteristics of the matrix • It should be non volatile • Less vapour pressure liquids Ex. Glycerol, Thioglycerol, 3-Nitrobengyl alcohol, Diethanolamine & Triethanolamine.
- 17. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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) - - - - - - - - - - - - - - - v vv v v v - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - v v v v v v v v + Sample soln 0.1 mg/ml Matrix soln 10 mg/ml Mixture soln Co-crystallization Crystallized sample-matrix mixture Ratio 1 : 1000 to 10000 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- 18. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 20 Then increasing the electric field on the droplet surface Solvent continues to evaporate Fine mist of charged droplets are created A nebulizer gas flows from outside the capillary to spray the sample Applying a high voltage about 3kv to 5kv ESI draws sample solution to the tip of capillary tube Electrospray ionization(ESI)
- 21. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 21 These gas phase ions enter into the mass analyser The droplets eventually become small enough that the sample ions are liberated into the gas phase As this evaporation and fission cycle is repeated Fission of molecules occurs It is softest ionization method , used for highly polar, least volatile/thermally unstable compounds. Ex. peptides, proteins, lipids, oligosaccharides.
- 22. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 22
- 23. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 23
- 24. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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 N2. 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)
- 25. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 25
- 26. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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 N2. Desolvation gas (heated N2) will be supplies which will convert the sprayed droplets into the form of vapours of analyte and solvent. Atmospheric pressure photoionization (APPI)
- 28. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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.
- 29. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 29
- 30. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 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. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 32