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Application Overview

High performance liquid chromatography-mass spectrometry (HPLC-MS, or more commonly LC-MS) is an extremely versatile analytical technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many applications because it offers very high sensitivity and specificity. Compared to GC-MS, the primary advantage of LC-MS is its capability to analyze a much wider range of analytes. Compounds that are thermally labile, exhibit high polarity or have a high molecular mass may all be analyzed using LC-MS.

The basic components of an HPLC system is described elsewhere: HPLC. A mass spectrometer has three main components: ionization source, mass analyzer, and detector (Figure 1).

Merck:/Freestyle/LW-Lab-Water/applications/LC-MS/LW-LC-LCMS-Image1-460x338.jpg
Figure 1: The main components of a mass spectrometer


Analytes getting into the mass spectrometer have to be converted to gaseous phase ions by the ion source. The two most common ion sources used for LC-MS are electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI):

Electrospray ionization (ESI) - Ions are generated at atmospheric pressure by passing the analyte solution through a capillary (electrospray needle) that has a high potential difference (with respect to the counter electrode) applied to it (typically between 2.5 to 4 kV). This initially produces aerosols of charged droplets that consist of both solvent and analyte molecules with a net positive or negative charge, depending on the polarity of voltage applied (hence the terms ESI positive and ESI negative). The analyte ions eventually become free of the solvent that surround them, and they are directed to the mass analyzer.

Atmospheric pressure chemical ionization (APCI) - Ions are generated at atmospheric pressure through ion/molecule reactions. The analyte solution is introduced through a capillary into a pneumatic nebulizer and desolvated in a heated quartz tube before interacting with the corona discharge from a very fine needle. Essentially, electrons from the corona discharge ionize reagent molecules (O2, N2, H2O) and solvent molecules that are present around the needle. A series of reactions eventually lead to efficient ionization of the analytes once they reach the discharge region. Analyte ions are then directed to the mass analyzer.

The mass analyzer in the mass spectrometer separates or sorts ions according to their mass-to-charge ratio (m/z). The most common mass analyzers in LC-MS are quadrupole, ion trap, and a combination of two mass analyzers (e.g. triple quadrupole, quadrupole time-of-flight, quadrupole linear ion trap) to carry out tandem mass spectrometry experiments (tandem MS). LC-MS/MS (also LC-MSn) offers further enhanced sensitivity and selectivity compared to LC-MS.

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