This document presents a seminar on ionization techniques in LC-MS (Liquid Chromatography-Mass Spectrometry) as part of the curricular requirements for a Master of Pharmacy program. It covers the integration of HPLC with mass spectrometry, various ionization methods such as Electron Ionization, Chemical Ionization, and Atmospheric Pressure Ionization, discussing their principles and applications. The material is intended to enhance understanding of analytical techniques and their relevance in pharmaceutical analysis.

1. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
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
Ionization Techniques in LC-MS

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
Hoffman and Stroobant “Mass spectroscopy principles and
applications” 3rd edition ,2007.
Agilent technologies “Basics of LC-MS”
References

33. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 33