Atmospheric Pressure Ionization (API) a Universal Interface for LC-MS

ByHammed Wasiu Adebayo

SGC100025Chemistry 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.

• 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.

Liquid chromatography-Mass spectrometry

• 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.

LC-MS an “odd couple”

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 ionizationWide 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

• 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.

A Quick look at the Earlier Interfaces (contd)

Figure 2: Thermospray interface. (a) configuration for ‘real-TSP-ionization’ (filament off) orexternal ionization (filament on). (b) configuration with discharge electrode for externalionization 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

• 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.

A “universal interface”

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.

Figure 3. Applications of various LC/MS ionization techniques

Ionization Techniques – Application range

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 Basics(Spraying a charged “mist”)

Electrospray ionization (ESI)

Figure 4. Electrospray ion source

High Voltage

Sample

Nebulizer Gas

charged droplets

++++ –

++–

+

+ –

+

++

+

++

+

++

+

++

+

+

Ion Source(atmosphere)

IonSpray inlet

Ions

To Q0(8x10 Torr)-3

Vacuum Interface

Turb

o G

as

~10,000,000 ions on column ~4,000,000 - 40,000 ionsOperator Impact Area

~1000 ions

• 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.

Electrospray ionization (ESI)

Figure 5. Desorption of ions from solution

• 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.

Electrospray ionization (ESI)(Facts & Applications)

– (“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 pressurechemical ionization (APCI)

Atmospheric pressurechemical 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

• 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.

Atmospheric pressure chemical ionization (APPI) (Application)

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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