Atmospheric Pressure Ionization (API)

a Universal Interface for LC-MS

By
Hammed Wasiu Adebayo
SGC100025
Chemistry Dept., University of Malaya
contents
• Introduction
• Mass spectrometer
• LC-MS “an odd couple”
• Earlier interfaces
• Disadvantages of earlier interfaces
• A universal interface
• Evolution of Atmospheric Pressure Ionization (API)
• ESI
• APCI
• APPI
• Putting the three together
• Summary
• Future development
• Reference
• acknowledgement
 Introduction
• Liquid chromatography is a fundamental
separation technique in the life sciences and
related fields of chemistry.
• Suitable for separation of non-volatile and
thermally fragile molecules from small-molecule
drug metabolites to peptides and proteins.
•  It has been said that over 80% of known organic
species are amenable to separation with liquid
chromatography.
 Mass spectrometers (MS)
• Mass spectrometers work by ionizing molecules and
then sorting and identifying the ions according to
their mass-to-charge (m/z) ratios.
• Mass spectrometry with its high sensitivity is capable
of providing structure, molecular weight, empirical
formula, and quantitative information about a
specific analyte.
Liquid chromatography-Mass spectrometry

•  LC-MS refers to the powerful combination of

liquid chromatographic separation with mass
spectrometric detection. 
• Gives ability to analyze virtually any molecular
species...thermally labile, non-volatile, and
high molecular weight species.
 LC-MS an “odd couple”
• Described by Patrick Arpino, LC-MS is like the bird and fish
courtship...love between totally incompatible species. 
• Mass spectrometry (the bird) is a powerful detection
technique for gas-phase ions. 
• Liquid Chromatography(the fish) is a liquid-phase separation
technique. 
• Liquids are incompatible with operating conditions of the
mass spectrometer, and gases are incompatible with the
operation of the liquid chromatography.
• Therefore, the interfacing between LC & MS is like the
"match-maker“ btw ALIENS & HUMAN BEINGS.
 Challenges in Interfacing LC to MS
Flow Rate Concerns
 Differential pumping can only handle 2 mL/min of
water.
 For maximum sensitivity, want to use all of the eluent.
Use of Buffers and Additives
 Non-volatile buffers a concern
 Some additives suppress ionization
Wide Range of Analytes
 Many are nonvolatile, thermally labile
 Attempts to solve the coupling problems

• Leads to development of various interfaces

Earlier Interfaces
• Direct Liquid Introduction
• Moving Belt
• Thermospray
• Particle Beam
• Continuous-Flow Fast Atom Bombardment
 A Quick look at the Earlier Interfaces

• Particle beam (PB) and moving belt rely on

removal of the solvent prior to entering the
MS.

Fig 1 principal component of a moving-belt interface

A Quick look at the Earlier Interfaces (contd)

• Coupling with continuous flow fast atom

bombardment (cf-FAB) or direct liquid
introduction (DLI) reduces the flow entering
the MS using some kind of splitting device.
A Quick look at the Earlier Interfaces (contd)

• Thermospray
liquid flow leaving an LC system is heated to
create a spray of superheated mist containing
small liquid droplets.
Ionization can be:
• Real thermospray that uses a volatile buffer
• One with an external ionization that uses
discharge electrode.
Figure 2: Thermospray interface. (a) configuration for ‘real-TSP-ionization’ (filament off) or
external ionization (filament on). (b) configuration with discharge electrode for external
ionization and repeller electrode. (Adapted from reference 14.)
 Main Disadvantages of Earlier LC/MS systems

 Inability to separate the mobile phase molecules from the

analyte molecules (direct liquid inlet, thermospray)
 Separation of the mobile phase molecules from the analyte
molecules before ionization (particle beam).
 Ionization takes place under vacuum by traditional electron
Ionization.(hard)
 Successful only for a very limited number of compounds.
 difficulty in operation
 Limited sensitivity
 Not robust
 Useful for specific applications
 A “universal interface”
• To solve the above mentioned problems

• Industry was desperate for a decent, rugged

LC/MS interface.
– GC/MS required derivatization, etc.
– Not applicable to most biomolecules (MW, etc.)
• Then API ionization ALONG WITH new
interface designs provided the solution.
Evolution of Atmospheric Pressure Ionization
(API)
With the introduction of atmospheric pressure
ionization (API) techniques:
• more compounds can be successfully
analyzed by LC/MS.
Because

API
. comes with better sensitivity and ruggedness
 Today’s LC/MS Ionization Methods
All Done at Atmospheric Pressure
• On-line techniques:
– Electrospray (ESI) - Fenn @ Yale ~1984
• Shared Nobel Prize in 2002 for this work with K. Tanaka
(MALDI) and K. Wüthrich (NMR)
– Atmospheric Pressure Chemical Ionization (APCI)
• Irabarne & Thomson ~1979
– Atmospheric Pressure Photo Ionization (APPI)
• Emerging, not as widely used yet.
• All of the above are done at atmospheric
pressure
– Significant change from traditional ionization
methods which were all done within the vacuum
chamber.
Ionization Techniques – Application range

Figure 3. Applications of various LC/MS

ionization techniques
 Electrospray Basics
(Spraying a charged “mist”)
How?
• The LC eluent is sprayed (nebulized) into a
chamber at atmospheric pressure in the
presence of a strong electrostatic field and
heated drying gas.
• The electrostatic field causes further
dissociation of the analyte molecules.
• The heated drying gas causes the solvent in
the droplets to evaporate.
 Electrospray ionization (ESI)
Vacuum Interface
IonSpray inlet
charged droplets

High Voltage
+ –

+ +
+
+
+++ + + To Q0
– ++ + +
++ – + + -3
+ + (8x10 Torr)
Sample +
+
Ions
as
oG
Nebulizer Gas
rb
Tu

Ion Source
(atmosphere)

~10,000,000 ions on column ~4,000,000 - 40,000 ions ~1000 ions

Operator Impact Area

Figure 4. Electrospray ion source

 Electrospray ionization (ESI)
• As the droplets shrink, the charge
concentration in the droplets increases making
the repulsive force between ions with like
charges exceeds the cohesive forces and ions
are ejected (desorbed) into the gas phase
yielding a very soft ionization technique.
• These ions are attracted to and pass through a
capillary sampling orifice into the mass
analyzer.

Figure 5. Desorption of ions from solution

 Electrospray ionization (ESI)
(Facts & Applications)
• ES is concentration-sensitive device rather than flow-rate of the analyte.
 This allows miniaturization of the technique without loss of
SENSITIVITY.
Useful for analyzing high molecular weight, thermolabile, polar
biomolecules...proteins, peptides, and oligonucleotides.
• Multiple charging makes electrospray useful to analyze molecules as
large as 150,000 u.
• Using deconvolution method a large molecule with many charges can be
resolved to determine the actual molecular weight of the analyte.
• A soft technique...can easily analyse a very LABILE structure without
structure distruption.
• Fragmentation can be induced using tandem MS to obtain structural
information.
 Atmospheric pressure
chemical ionization (APCI)
– (“Steam distill” LC eluent past a HV needle)
– APCI utilizes corona discharge
– APCI is a “three” step process:
• 1) Needle at high voltage ionizes nebulizing gas
(air or nitrogen) forming primary ions.
• 2) Primary ions react immediately with solvent
molecules forming reagent ions
• 3) Reagent ions react (by proton transfer) with
analyte molecules forming (M+H)+ in positive ion
mode or (M-H)- in negative ion mode.
 Atmospheric pressure
chemical ionization (APCI)

Figure 4. APCI ion source

Atmospheric pressure chemical ionization (APCI)
(Facts & Applications)
• Better ionization efficiency compares to CI because of atmospheric
pressure.
• APCI is applicable to a wide range of polar and nonpolar
molecules.
• Typically used for molecules less than 1,500 amu because it rarely
results in multiple charging.
• Compared with ESI, APCI is less well-suited for analysis of large
biomolecules that may be thermally unstable. because it involves
high temperatures. Perform better at high flow-rates unlike ESI
suited to minoaturization.
• APCI is used with normal-phase chromatography more often than
electrospray is because the analytes are usually nonpolar.
 Atmospheric pressure photoionization
(APPI)
• A relatively new technique.
• As in APCI, a vaporizer converts the LC eluent to the
gas phase. A discharge lamp (UV lamp) generates
photons in a narrow range of ionization energies.
• The range of energies is carefully chosen to ionize as
many analyte molecules as possible while minimizing
the ionization of solvent molecules.
• The resulting ions pass through a capillary sampling
orifice into the mass analyzer.
Atmospheric pressure photoionization
(APPI)

Figure 5. APPI ion source

Atmospheric pressure chemical ionization (APPI)
(Application)
• Applicable to many of the same compounds
that are typically analyzed by APCI.
• It shows particular promise in two
applications:
i. highly nonpolar compounds and
ii. low flow rates (<100 μl/min).
APCI demonstrates reduced sensitivity in the
cases above.
 Putting the three together
• The nature of the analyte(s) and the
separation conditions have a strong influence
on which ionization technique: electrospray,
APCI, or APPI, will generate the best results.
• The most effective technique is not always
easy to predict.
• ES and APCI are complementary techniques
 Future Developments
• As sample availability and sensitivity are always an issue in
analytical (bio)chemistry, miniaturization will be a continuing
trend in LC–MS.
• Miniaturization of the separation techniques and consequent
development of the appropriate interfaces will proceed, including
chip-based technology for both separation and interfacing to MS.
• Off-line techniques, e.g, MALDI, 2D gel electrophoresis etc., will
be modified and new techniques developed to couple with
existing MS and LC–MS systems.
• FOR LC-MS developers, API remains the beginning of a NEW
REVOLUTION!
 summary

1. “API approaches can handle volumes of liquid typically

used in LC”
2. “API is suitable for the analysis of nonvolatile, polar and
thermally unstable compounds typically analysed by LC”
3. “API-MS systems are sensitive, offering comparable or
better detection limits than achieved by GC–MS”
4. “API systems are very rugged and relatively easy to use.”
5. Miniaturization is one of the future development of API
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