High Performance Liquid Chromatography Mass Spectrometry (HPLC/MS)

High performance liquid chromatography mass spectrometry is an extemely versatile instrumental technique whose roots lie in the application of more traditional liquid chromatography to theories and instrumentation that were originally developed for gas chromatography (GC). As the name suggest the instrumentation comprises a high performance liquid chromatograph (HPLC) attached, via a suitable interface, to a mass spectrometer (MS). The primary advantage HPLC/MS has over GC/MS is that it is capable of analysing a much wider range of components. Compounds that are thermally labile, exhibit high polarity or have a high molecular mass may all be analysed using HPLC/MS, even proteins may be routinely analysed. Solutions derived from samples of interest are injected onto an HPLC column that comprises a narrow stainless steel tube (usually 150 mm length and 2 mm internal diameter, or smaller) packed with fine, chemically modified silica particles. Compounds are separated on the basis of their relative interaction with the chemical coating of these particles (stationary phase) and the solvent eluting througn the column (mobile phase). Components eluting from the chromatographic column are then introduced to the mass spectrometer via a specialised interface. The two most common interfaces used for HPLC/MS are the electrospray ionisation and the atmospheric pressure chemical ionisation interfaces.

Electrospray ionisation

In electrospray ionisation the analyte is introduced to the source at  flow rates  typically of the order of 1µl min-1. The analyte solution flow passes through the electrospray needle that has a high potential difference (with respect to the counter electrode) applied to it (typically in the range from 2.5 to 4 kV). This forces the spraying of charged droplets from the needle with a surface charge of the same polarity to the charge on the needle. The droplets are repelled from the needle towards the source sampling cone on the counter electrode (shown in blue). As the droplets traverse the space between the needle tip and the cone, solvent evaporation occurs. This is circled on the Fig.1 and enlarged upon in Fig.2. As the solvent evaporation occurs, the droplet shrinks until it reaches the point that the surface tension can no longer sustain the charge (the Rayleigh limit) at which point a "Coulombic explosion" occurs and the droplet is ripped apart. This produces smaller droplets that can repeat the process as well as naked charged analyte molecules. These charged analyte molecules (they are not strictly ions) can be singly or multiply charged. This is a very soft method of ionisation as very little residual energy is retained by the analyte upon ionisation. It is the generation of multiply charged molecules that enables high molecular weight components such as proteins to be analysed since the mass range of the mass spectrometer is greatly increased since it actually measures the mass to charge ratio rather than mass per se. The major disadvantage of the technique is that very little (usually no) fragmentation is produced although this may be overcome through the use of tandem mass spectrometric techniques such as MS/MS or MSn.

Schematic of an ESI interface
Figure 1  A schematic of an ESI interface

Closer schematic of an ESI interface

Figure 2  A schematic of the mechanism of ion formation

Atmospheric pressure chemical ionisation

Atmospheric pressure chemical ionisation (APCI) is an analogous ionisation method to chemical ionisation (CI). The significant difference is that APCI occurs at atmospheric pressure and has its primary applications in the areas of ionisation of low mass compounds (APCI is not suitable for the analysis of thermally labile compounds). The general source set-up (see Fig. 3) shares a strong resemblance to ESI. Where APCI differs to ESI, is in the way ionisation occurs. In ESI, ionisation is bought about through the potential difference between the spray needle and the cone along with rapid but gentle desolvation. In APCI, the analyte solution is introduced into a pneumatic nebulizer and desolvated in a heated quartz tube before interacting with the corona discharge creating ions. The corona discharge replaces the electron filament in CI - the atmospheric pressure would quickly "burn out" any filaments - and produces primary N2°+ and N4°+ by electron ionisation. These primary ions collide with the vaporized solvent molecules to form secondary reactant gas ions - e.g. H3O+ and (H2O)nH+ (see Fig. 4). These reactant gas ions then undergo repeated collisions with the analyte resulting in the formation of analyte ions. The high frequency of collisions results in a high ionisation efficiency and thermalisation of the analyte ions. This results in spectra of predominantly molecular species and adduct ions with very little fragmentation. Once the ions are formed, they enter the pumping and focussing stage in much the same as ESI.

Schematic of an APCI interface
Figure 3  A schematic of the components of an APCI source

Closer schematic of an APCI interface

Figure 4  A more detailed view of the mechanism of APCI

Diagrams and text (partial) by Dr Paul Gates, School of Chemistry, University of Bristol