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

By Hammed Wasiu Adebayo SGC100025 Chemistry Dept., University of Malaya

‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ 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

‡ It has been said that over 80% of known organic species are amenable to separation with liquid chromatography. .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.

empirical formula. and quantitative information about a specific analyte. .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. molecular weight. ‡ Mass spectrometry with its high sensitivity is capable of providing structure.

.thermally labile.. ‡ Gives ability to analyze virtually any molecular species. and high molecular weight species.Liquid chromatography-Mass spectrometry ‡ LC-MS refers to the powerful combination of liquid chromatographic separation with mass spectrometric detection. non-volatile. .

and gases are incompatible with the operation of the liquid chromatography.LC-MS an odd couple ‡ Described by Patrick Arpino. ‡ Liquids are incompatible with operating conditions of the mass between totally incompatible species. LC-MS is like the bird and fish courtship. .. the interfacing between LC & MS is like the "match-maker btw ALIENS & HUMAN BEINGS.. ‡ Therefore. ‡ Liquid Chromatography(the fish) is a liquid-phase separation technique. ‡ Mass spectrometry (the bird) is a powerful detection technique for gas-phase ions.

Challenges in Interfacing LC to MS      Flow Rate Concerns Differential pumping can only handle 2 mL/min of water. want to use all of the eluent. thermally labile . For maximum sensitivity. Use of Buffers and Additives Non-volatile buffers a concern Some additives suppress ionization Wide Range of Analytes Many are nonvolatile.

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 .

Fig 1 principal component of a moving-belt interface .A Quick look at the Earlier Interfaces ‡ Particle beam (PB) and moving belt rely on removal of the solvent prior to entering the MS.

.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.

. 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) ‡ Thermospray liquid flow leaving an LC system is heated to create a spray of superheated mist containing small liquid droplets.

(b) configuration with discharge electrode for external ionization and repeller electrode. (a) configuration for real-TSP-ionization (filament off) or external ionization (filament on). (Adapted from reference 14.Figure 2: Thermospray interface.) .

Ionization takes lace nder vac m y traditional electron Ionization.( ard) S ccessf l only for a very limited n m er of com o nds. diffic lty in o eration Limited sensitivity ot ro st sef l for s ecific a lications . t ermos ray) Se aration of t e mo ile ase molec les from t e analyte molec les efore ionization ( article eam).Main Disadvantages of Earlier LC/MS systems Ina ility to se arate t e mo ile ase molec les from t e analyte molec les (direct li id inlet.

± GC/MS required derivatization. rugged LC/MS interface. etc.) ‡ Then API ionization ALONG WITH new interface designs provided the solution. ± Not applicable to most biomolecules (MW. etc. .A universal interface ‡ To solve the above mentioned problems ‡ Industry was desperate for a decent.

Because API comes with better sensitivity and ruggedness .Evolution of Atmospheric Pressure Ionization (API) With the introduction of atmospheric pressure ionization (API) techniques: ‡ more compounds can be successfully analyzed by LC/MS. .

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

Ionization Techniques Application range Figure 3. Applications of various LC/MS ionization techniques .

‡ The heated drying gas causes the solvent in the droplets to evaporate.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. .

Electrospray ionization (ESI) IonSpray inlet charged droplets Vacuum Interface High Voltage + ² + ++++ ² + ++ ² + + + + + + + + + + + + + Sample To Q0 -3 (8x10 Torr) Ions Nebulizer Gas Ion Source (atmosphere) ~10. Electrospray ion source .000 ions Operator Impact Area ~1000 ions Figure 4.000.000 ions on column ~4.000.000 .40.

Desorption of ions from solution .Electrospray ionization (ESI) ‡ As the droplets shrink. Figure 5. ‡ These ions are attracted to and pass through a capillary sampling orifice into the mass analyzer. 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.

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