Electrospray ionisation

John Fenn was also recognised with the Nobel Prize in Chemistry in 2002, for his work on electrospray ionisation (ESI). He demonstrated that multiply charged ions could be obtained from proteins up to 130 kiloDaltons (kDa) in molecular weight using ESI. Unlike MALDI, which is a pulsed ionisation process performed under vacuum; ESI takes place at atmospheric pressure and is a continuous ionisation process.

Figure 1.3 The ESI ionisation process.

6 For ESI, a sample is normally prepared in aqueous solution containing organic solvent such as acetonitrile (ACN) or methanol (MeOH). For positive ion analysis, a small amount of acid (formic or acetic) is commonly added to the solution to aid the ionisation process. This solution is sprayed through a capillary needle to which a high electric potential is applied. The most commonly accepted mechanisms by which ion formation takes place in ESI is represented by Figure 1.3.

The charged solution inside the capillary forms a Taylor cone at the end of the needle, which releases small highly charged droplets. The formation of these droplets is aided by a nebulising gas, such as nitrogen. Two mechanisms have been proposed that describe the formation of ions by ESI. These are the ion evaporation model (IEM) and the charge residue model (CRM). In both models charge repulsion in the droplets exceeds the surface tension causing solvent molecules to evaporate. The charge repulsion causes a Coulombic explosion and the droplets divide. In CRM this process is continuously repeated until a single, multiply-charged analyte ion is produced. For multiply charged ions at high m/z ratio, CRM is thought to be the dominant mechanism. In contrast, IEM is thought to be the model by which ions of low m/z are formed. The intial process is the same, but following the Coulombic explosion ions within the newly formed droplets are desorbed into the gas-phase and subsequently enter the mass spectrometer (Beaudry, Le Blanc et al. 1999).

The continuous ionisation offered by ES enables it to be routinely coupled to high performance liquid chromatography (HPLC) systems which are employed to separate biomolecules prior to MS analysis.

1.1.3 Mass analysers

Gas-phase ions formed during the ionisation process are subsequently separated according to their mass-to-charge (m/z) ratio. There are a number of mass analysers used in modern mass spectrometry, each with their own advantages and limitations. The mass analyser of choice should be considered based on the application for which it is to be used, taking into account requirements for resolving power, mass range and transmission efficiency.

7 Resolving power may be defined as the ability to separate two ions of near m/z value. For the purposes of this thesis, resolution will be defined by full width at half maximum (FWHM). This is based on a single well-resolved peak according to the equation:

Equation 1.1 M is the mass associated with the apex of the peak, and ΔMx is the width of the peak

at a specified height, here taken to be 50% (half-maximum).

1.1.3.1 Quadrupole

A quadrupole mass analyser consists of four parallel metal rods. A combination of radio frequency (RF) and direct current (DC) voltages are applied to opposite pairs of rods, which allow the quadrupole to operate as a mass filter. The principle of the quadrupole was introduced by Paul and Steinwedel (Paul and Steinwedel 1953). Quadrupoles are low-resolution mass analysers that operate at unit mass resolution. They are often combined in series or with other forms of mass analysers, allowing them to be used for targeted analysis of specific compounds, greatly increasing the selectivity of the experiment.

8 A combination of RF and DC voltages applied to the rods affect the trajectory of ions travelling through the quadrupole and only allow ions of a particular m/z to pass through with a stable trajectory. All other ions will collide with the rods or the sides of the mass analyser and will be lost. When operated in RF-only mode, the quadrupole operates as an ion guide allowing ions to pass through to reach the detector.

1.1.3.2 Time-of-flight

Time-of-flight (TOF) mass spectrometry was first described by Stephens in 1946. He outlined a new type of mass spectrometer was under construction that “should be well suited for gas composition control, rapid analysis and portable use” (Stephens 1946). The first commercial TOF instrument was based on a design published in 1955 by Wiley and McLaren (Wiley and McLaren 1955). TOF analysers are compatible with the pulsed ionisation of MALDI, and offer rapid analysis, a wide m/z range, and theoretically no upper mass limit. TOF mass analysers also have very high transmission efficiency and therefore high sensitivity.

Following ionisation ions are accelerated by an electric field. The ions then enter a free-field region, known as a flight tube. Here the ions are separated according to their velocity, which depends on the m/z ratio of the ion. The time each ion takes to traverse the flight tube is recorded by the detector; ions with smaller m/z move faster through the flight tube than those with a larger m/z. This can be described using the following equation, where t = time of flight, d = distance, m = mass, eV = accelerating voltage and z = charge.

(

)

Equation 1.2 The mass resolution of TOF analysers is proportional to the flight path and time and poor resolution proved to be a major drawback with initial TOF analysers. This was in part due to a spread of kinetic energy amongst ions with the same m/z, caused by a number of factors including time, space and kinetic energy distribution. This kinetic energy spread results in peak broadening which, in turn, decreases resolution. The

9 introduction of delayed pulsed extraction and the reflectron, markedly improved the achievable resolution of TOF analysers.

When using delayed pulse extraction, in order to reduce the kinetic energy spread amongst ions with the same m/z, a time delay is introduced between ion formation and extraction. This technique was first considered by Wiley and McLaren and termed time-lag focusing. Delayed pulse extraction allows ions to separate according to kinetic energy following ion formation. An extraction voltage pulse is then applied to extract the ions from the source after a delay. This corrects the kinetic energy spread, thereby improving the TOF resolution. Both pulse and delay can be adjusted for the mass range of the ions of interest, to provide optimal resolution. A reflectron design, proposed by Mamyrin in 1973 (Mamyrin, Karataev et al. 1973), can also be used to improve the resolution in a TOF analyser. This is an electrostatic reflector which acts as an ion mirror to deflect ions back through the flight tube. Reflectrons also correct the kinetic energy spread amongst ions of the same m/z, as ions with higher kinetic energy penetrate the reflectron more deeply than those with lower kinetic energy. This is outlined in Figure 1.5.

Figure 1.5 Schematic representation of a reflectron TOF instrument.

Two ions of equal m/z, but different initial kinetic energies, will both arrive at the detector at the same time. The ion with high kinetic energy goes deeper into the reflectron than the ion with low kinetic energy.

10 Ions with higher energy will therefore spend longer in the reflectron and both ions with the same m/z will reach the detector at the same time thereby reducing peak broadening. Whilst there is an improvement in resolution, mass range can be limited and sensitivity reduced when using a reflectron-TOF analyser. Dual-stage reflectrons may be used to improve the reflectron performance; here the first reflectron has a higher electric field to decelerate the ions and the second has a weaker field. Dual- stage reflectrons may also suffer from lower transmission efficiency, but can correct a wider range of kinetic energy spread and provide higher mass resolution (de Hoffmann and Stroobant 2009).

The development of orthogonal acceleration (oa) in TOF analysers has, to an extent, overcome the difficulties in combining TOF with continuous ionisation sources (e.g. ESI) that do not produce packets of ions. Orthogonal acceleration was first proposed in 1964 (O'Halloran, Fluegge et al. 1964), but its full potential was not realised until much later with the work of Guilhaus and Dodonov (Dawson and Guilhaus 1989; Coles and Guilhaus 1993). In an oa-TOF instrument, the ion source generates an ion beam that is directed into an orthogonal accelerator region, perpendicular to the TOF. An electrostatic field is created by applying a large voltage pulse, and a packet of ions is pushed at a right-angle away from the ion beam and into the strong acceleration field towards the TOF. This is shown in Figure 1.6.

When using reflectron TOF analysers, the detector can be conveniently located next to the orthogonal accelerator for improved resolution. The introduction of oa-TOF analysers has allowed ESI applications to benefit from the high mass range, speed, sensitivity and mass accuracy of TOF analysers (Guilhaus, Selby et al. 2000; McLuckey and Wells 2001).

11

Figure 1.6 Simplified schematic representation of an oa-TOF reflectron system. The ion beam enters from a source at the left and enters the orthogonal accelerator (oa). A voltage pulse is then applied, perpendicular to the ion beam, which sends a packet of ions into the accelerator region and towards the TOF. Here a single-stage reflectron is represented. Whilst the ion packet traverses the TOF the oa refills with the next packet of ions. Adapted from (Guilhaus, Selby et al. 2000).

12

1.1.3.3 Ion Trap

Ion trap analysers use an oscillating electric field to store ions, which are then ejected from the trap according to their m/z values in order to create the spectrum. Two types of ion trap exist, the 2D or linear ion trap and the 3D ion trap. Paul and Steinwedel had patented an ion trap instrument in 1960 (Paul and Steinwedel 1960), but it was not until Stafford et al. modified the design that ion traps were used more widely (Stafford, Kelley et al. 1984).

The 3D ion trap consists of a circular electrode, with ellipsoid caps on either end. Ions are pulsed into the trapping region, where they are held in a 3D trajectory by overlapping DC and RF voltages. Ions of different masses are contained in the trap and released based on their m/z. With many ions present in the trap, they may repel each other causing an expansion of their trajectories. This is known as space charging and can result in ion losses. To remove excess energy from the ions, the trap is maintained at pressure using helium gas, which collides with the ions and prevents losses from the trap. The number of ions within the trap is maintained by a gating lens; too few ions may cause a loss of sensitivity, whilst too many may cause a loss of resolution through the introduction of space charge effects.

2D or linear ion traps (LIT) are similar in principle to the quadrupole mass analyser, consisting of four parallel rods with lenses at each end. These lenses repel the ions and trap them inside the rods. LITs have an advantage over 3D ion traps in that they have a much higher trapping capacity, which reduces the space-charge effect. Ions are selectively ejected from the trap either perpendicularly to or along the axis of the trap (de Hoffmann and Stroobant 2009).

1.1.3.4 Orbitrap

In June 2005 a new mass analyser, known as the Orbitrap, was introduced by the Thermo Electron Corporation. This was based on a concept initially proposed by Makarov in 2000, which involved trapping ions within an electrostatic field. Mass spectra are obtained using a Fourier Transform (FT) of the induced ion current (Makarov 2000). Ions orbit around a central axial electrode, in the absence of either magnetic or RF fields. These orbiting ions perform harmonic oscillations along this

13 electrode at a frequency proportional to (m/z)-½ and independent of energy and spatial spread. These oscillations are detected using image current detection before conversion into mass spectra using FT (Hu, Noll et al. 2005).

The Orbitrap can be characterised as a high-resolution and high mass accuracy instrument; though it should be noted that Orbitrap resolution is dependent both on m/z and scan time. The resolution is currently specified at 60,000 (FWHM) at m/z 400 with a scan time of 1 second. This increases linearly with scan time and can reach over 100,000. The achievable resolution is inversely proportional to (m/z) -½, and so decreases as the mass range is increased (de Hoffmann and Stroobant 2009).