Atmospheric pressure ionization (API) techniques allow ionization of analytes at atmospheric pressure for mass spectrometry analysis. The three main API techniques discussed are electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI). ESI uses an electric field to generate charged droplets and gas phase ions. APCI uses the solvent as a reagent gas for chemical ionization of analytes. APPI uses ultraviolet light to ionize analytes or dopants which then undergo gas phase reactions. These API techniques revolutionized liquid chromatography-mass spectrometry by enabling ionization of biomolecules at atmospheric pressure for analysis.

1. Atmospheric Pressure IonizationAtmospheric Pressure Ionization
(API)(API)
Presented By:
Vinitha Nair
M.Sc Bioanalytical Sciences Part 2

2. What is MS?
An instrument that separates molecules orAn instrument that separates molecules or
atoms based on their mass/charge ratioatoms based on their mass/charge ratio
(m/z).(m/z).
– In order to control them, you need to put a
charge on them.
– In order to separate them, you need an
“analyzer”

4. Why LC/MS?
GC/MS required
derivatization, etc.
Not applicable to
most
biomolecules
(MW, etc.)
Industry wasIndustry was
desperatedesperate
for a decent,for a decent,
ruggedrugged
LC/MSLC/MS
interface.interface.
Because

5. Why API?
 Revolutionized LC/MS
 Desolvation and/or ionization of analytes
occurs at atmospheric pressures
 Gas phase ions are sampled by the high
vacuum mass spectrometer.

6. Ion Source Interface Mass Analyzers Detection
-ESI
-APCI
-APPI
LC
MS System – under vacuum
Block Diagram of LCMS

7. API
SourceAll the techniques are done at
atmospheric pressure
Significant change from traditional
ionization methods which were all done
within the vacuum chamber.

8. Types of API Sources

9. Ionization process which uses electrical fields to generate charged droplets and
subsequent analyte ions by ion evaporation for MS analysis.

10. Gas
Nebulized
Spray
LC
Heater
ESI Key Features:
- Nebulized flow through capillary
- Heat applied to evaporate solvent
- Capillary (LC) 90o
to Cone (MS)
Sample
Cone
MS
CapillaryIonization
Chamber
760 torr
10-6
torr
- Atmospheric pressure ion source
- Voltage difference applied between
capillary and cone
-
+
- Detect (+) ions  Cap (+), Cone (-)
- Detect (-) ions  Cap (-), Cone (+)

13. A gas phase chemical ionization (CI) process where the solvent acts as the CI
reagent gas to ionize the sample.

14. Gas
Nebulized
Spray
LC
Heater
APCI Key Features:
- Nebulized flow through capillary
- Heat applied to evaporate solvent
- Capillary (LC) 90o
to Cone (MS)
MS
CapillaryIonization
Chamber
760 torr
10-6
torr
- Atmospheric pressure ion source
- No voltage applied to capillary
- droplets are not charged
-
- Voltage still applied to cone
- Detect (-) ions  Cone (+)
- Detect (+) ions Cone (-)
Sample
Cone
- Heat evaporates solvent, analytes
partition into the gas-phase
uncharged
Corona
discharge
needle

16. The protonated analyte ions are then formed by gas-phase ion-molecule reactionsThe protonated analyte ions are then formed by gas-phase ion-molecule reactions
of these charger cluster ions with the analyte molecules. This results in theof these charger cluster ions with the analyte molecules. This results in the
abundant formation of [M+H]abundant formation of [M+H]++
ions.ions.

17. Krypton lamp producing ultraviolet light ionizes gas phase analytes or dopants
with subsequent gas-phase reactions.

18. Gas
Nebulized
Spray
LC
Heater
APPI Key Features (same as APCI):
- Nebulized flow through capillary
- Heat applied to evaporate solvent
- Capillary (LC) 90o
to Cone (MS)
MS
CapillaryIonization
Chamber
760 torr
10-6
torr
- Atmospheric pressure ion source
- No voltage applied to capillary
- droplets are not charged
-
- Voltage still applied to cone
- Detect (-) ions  Cone (+)
- Detect (+) ions Cone (-)
Sample
Cone
- Heat evaporates solvent, analytes
partition into the gas-phase
uncharged
UV
lamp

20. Direct APPIDirect APPI
M + hν ⇨ M+•
+ e-
M + S + hν [M + H]⇨ +
+ [S - H]-
(solvent dependent)
A minority of them will be ionized directly by the UV light (photoionization).
Some of the analyte molecules can be indirectly ionized with the help of the solvent
molecules.
You end up with two types of ions (M+•
and [M + H]+
) from one compound, which then
proceed to the MS to be analyzed.

21. Dopant-Assisted APPIDopant-Assisted APPI
D+•
+ M [M + H]⇨ +
+ [D - H]•
D+•
+ M M⇨ +•
+ D
The dopant ion can donate a proton to the analyte molecule.
The dopant ion receives an electron from the analyte molecule.
The result is an ionized sample molecule.

22. Relative Applicability of API Techniques
MolecularWeight

23. References:
•usd-chem730.wikispaces.com/file/view/ESI-MS.ppt
•ccc.chem.pitt.edu/wipf/Agilent%20LC-MS%20primer.pdf
• link.springer.com/article/10.1007%2FBF02493125
•ccc.chem.pitt.edu/wipf/Agilent%20LC-MS%20primer.pdf
•137.122.232.177/NRL%20publications/2007HarrisPhytochem.pdf
•usd-chem730.wikispaces.com/file/view/ESI-MS.ppt
•ccc.chem.pitt.edu/wipf/Agilent%20LC-MS%20primer.pdf
• link.springer.com/article/10.1007%2FBF02493125
•ccc.chem.pitt.edu/wipf/Agilent%20LC-MS%20primer.pdf
•137.122.232.177/NRL%20publications/2007HarrisPhytochem.pdf
•Principles & Practices of Bio-analysis – Richard Venn•Principles & Practices of Bio-analysis – Richard Venn
Bibliography
Webliography