Mass spectrometry (MS)_Part 2

Methods of sample ionisation

Many ionisation methods are available and each has its own advantages and disadvantages ("Ionization Methods in Organic Mass Spectrometry", Alison E. Ashcroft, The Royal Society of Chemistry, UK, 1997; and references cited therein).

The ionisation method to be used should depend on the type of sample under investigation and the mass spectrometer available.

Ionisation methods include the following:
Atmospheric Pressure Chemical Ionisation (APCI)
Chemical Ionisation (CI)
Electron Impact (EI)
Electrospray Ionisation (ESI)
Fast Atom Bombardment (FAB)
Field Desorption / Field Ionisation (FD/FI)
Matrix Assisted Laser Desorption Ionisation (MALDI)
Thermospray Ionisation (TSP)

The ionisation methods used for the majority of biochemical analyses are Electrospray Ionisation (ESI) and Matrix Assisted Laser Desorption Ionisation (MALDI) , and these are described in more detail in Sections 5 and 6 respectively.

With most ionisation methods there is the possibility of creating both positively and negatively charged sample ions, depending on the proton affinity of the sample. Before embarking on an analysis, the user must decide whether to detect the positively or negatively charged ions

Electrospray ionisation

Electrospray ionisation
Electrospray Ionisation (ESI) is one of the Atmospheric Pressure Ionisation (API) techniques and is well-suited to the analysis of polar molecules ranging from less than 100 Da to more than 1,000,000 Da in molecular mass.

A suitable ESI source for the mass-spectrometric analysis was designed by the Fenn group in the mid 1980s .Later on it was modified by different research groups to improve the system’s robustness. Generally a dilute (less than mM in polar volatile solvent) analyte solution is injected by a mechanical syringe pump through a hypodermic needle or stainless steel capillary (~0.2 mm o.d and ~0.1 mm i.d) at low flow rate (typically 1–20 μL/min). A very high voltage (2–6 kV) is applied to the tip of the metal capillary relative to the surrounding source-sampling cone or heated capillary (typically located at 1–3 cm from the spray needle tip). This strong electric field causes the dispersion of the sample solution into an aerosol of highly charged electrospray (ES) droplets . A coaxial sheath gas (dry N₂) flow around the capillary results in better nebulization. This gas flow also helps to direct the spray emerging from the capillary tip towards the mass spectrometer. The charged droplets diminish in size by solvent evaporation, assisted by the flow of nitrogen (drying gas).

Finally the charged analytes are released from the droplets, some of which pass through a sampling cone or the orifice of a heated capillary (kept in the interface of atmospheric pressure and the high vacuum) into the analyser of the mass spectrometer, which is held under high vacuum. The heated capillary (typically 0.2 mm inner diameter, 60 mm in length and heated to 100–300°C) causes the complete desolvation of the ions passing through it. The use of drying gas and the heated capillary can influence the system’s robustness and reduce the degree of cluster ion formation . The transfer of analyte ions from solution to gas phase is not an energetic process, but rather the desolvation process effectively cools the gaseous ions. So the analyte ions with low internal energies are allowed to enter into the mass spectrometer from the electrospray probe, and the structure of the analytes generally remain intact (no fragmentation) when appropriate instrumental conditions (e.g., no activation of the ions in gas phase) are used. Nowadays a number of sprayer modifications like pneumatically assisted electrospray , ultrasonic nebulizer electrospray , electrosonic spray , and nanoelectrospray have been developed to expand the range of ESI applications. Among them the most popular one is nanoelectrospray.

After the development of ESI-MS, many different assumptions and hypotheses were made in the early 1990s to interpret the multiple charging of the analyte by the ES process . That time it was thought that the distribution of the charge states in the ESI-MS spectra actually reflects the degree of charging of the analyte (say proteins) in the neutral solution (as determined by solution phase equilibrium). But later it became evident that there is no correlation between solution charging and electrospray charging after the report of Kelly and coworkers. Their report showed that in positive ion mode ESI produced protonated (positively charged) analyte (myoglobin) though the analyte is overall negatively charged (deprotonated) in basic aspirating solution (pH 10), and in negative ion mode it produced deprotonated (negatively charged) analyte (myoglobin) though the analyte is overall positively charged (protonated) in acidic aspirating solution (pH 3). These observations implied that the charging process in ESI might occur in an entirely different manner than that it was thought.

When an analyte is transferred from solution to the gas phase via ESI, the analyte solution undergoes three major processes. These are (a) production of the charged droplets from the high-voltage capillary tip where the analyte solution is injected; (b) repeated solvent evaporation (from the charged droplet) and droplet disintegration, resulting a very small charged droplet, which is able to produce the charged analyte; (c) finally a mechanism by which the gas-phase ion is formed. Since the electrospray process existed long before its application in the mass spectrometry and earlier it was used for the electrostatic dispersion of liquids and the creation of aerosol, the first two processes were mostly studied by the researchers in the aerosol science, and the processes are well understood nowadays . But the last process, that is, the mechanisms of the ion formation from vary small highly charged droplet is still under controversy, and the exact process happening at this stage is not unambiguously known. Over the last two decades, the issue has hotly been discussed and debated, and several hypotheses based on the theory and experimental evidences were invoked , and here we would discuss the most popular models of the gas-phase ion formation

During standard electrospray ionisation the sample is dissolved in a polar, volatile solvent and pumped through a narrow, stainless steel capillary (75 - 150 micrometers i.d.) at a flow rate of between 1 �L/min and 1 mL/min. A high voltage of 3 or 4 kV is applied to the tip of the capillary, which is situated within the ionisation source of the mass spectrometer, and as a consequence of this strong electric field, the sample emerging from the tip is dispersed into an aerosol of highly charged droplets, a process that is aided by a co-axially introduced nebulising gas flowing around the outside of the capillary. This gas, usually nitrogen, helps to direct the spray emerging from the capillary tip towards the mass spectrometer. The charged droplets diminish in size by solvent evaporation, assisted by a warm flow of nitrogen known as the drying gas which passes across the front of the ionisation source. Eventually charged sample ions, free from solvent, are released from the droplets, some of which pass through a sampling cone or orifice into an intermediate vacuum region, and from there through a small aperture into the analyser of the mass spectrometer, which is held under high vacuum. The lens voltages are optimised individually for each sample.