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Ion-mobility-mass spectrometry (IM-MS) is an analytical technique whereby the conformation of molecules can be studied. It was first introduced in the 1970’s under the name plasma chromatography as a method to separate ions (Cohen and Karasek, 1970) and was soon recognised as a powerful tool that could be used to analyse a whole host of molecules (Collins and Lee, 2001). During the 1990’s the technique was applied to larger biomolecules with Clemmer demonstrating protein conformer separation using cytochrome c (Clemmer et al., 1995). The development of high resolution IM-MS over the past decade has further

89 enabled the separation of structural isomers that have an identical mass

(Wu et al., 2000).

As described previously conventional mass spectrometry involves four processes: sample introduction, sample ionisation, mass separation and detection. The addition of an IM cell to a mass spectrometer allows for an extra separation step which is dependent on the mobility of the ions (Kanu et al., 2008). The mobility of an ion is measured by the time it takes to pass through a buffer gas which is subjected to a weak electric field (drift time). The mobility can be dependent on many factors including molecular mass, charge on the ion, size of the ion and the polarizability of the drift gas used (Karasek, 1974).

4.2.2 TWIM-MS

Four types of IM-MS are available but only one will be discussed here: TWIM-MS (Pringle et al., 2007). Unlike other traditional IM-MS methods where the electric field is applied constantly, a high electric field is applied to one part of the cell and is swept sequentially through the cell in the direction that the ions are moving. This is achieved by using a stacked ring ion guide (SRIG) (Giles et al., 2004) which consists of a series of ring electrodes stacked orthogonally to the axis on which the ions travel. Ions are propelled down the SRIG in pulses or “waves” and it is therefore given the name “travelling wave”. The ions exit the IM cell at unequal times because the time it takes for the ion to exit the cell is dependent on its mobility through an inert gas as it travels along the travelling voltage

90 wave. Separation of ions is then achieved as ions that have a high mobility can keep up with the wave and exit the IM cell faster. Ions that have a lower mobility have more interactions with the buffer gas, roll over the top of the wave and have to wait for ensuing waves to propel them through the cell. As a consequence these ions exit the cell later (Giles et al., 2004). A longer drift time through the IM cell indicates that the ion has a larger collisional cross section (Jarrold, 1999). The quantity of ions exiting the cell is plotted as a function of time, known as the arrival time distribution (ATD).

4.2.3 A commercially available TWIM-MS instrument: Synapt HDMS The travelling wave ion guide has been integrated into a conventional hybrid Q-TOF mass spectrometer to produce the Synapt HDMS instrument which is manufactured by Waters (Pringle et al., 2007). The IM cell is located between two mass analysers, namely a quadrupole and a TOF. A close-up view of the TriWave cell is shown in Figure 4.1. The IM cell contains three travelling wave-enabled SRIGs: trap, IM and transfer cell. Ions first accumulate in the trap, are released into the IM ion guide for the actual separation and then pass into the transfer region to allow them to travel to the TOF mass analyser (Pringle et al., 2007). Although suggested flow rates for the buffer gas in this region are indicated on the diagram, these can be optimised depending on the sample. The presence of a helium cell prior to the nitrogen region maximises the transmission of the ions into the IM cell.

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Figure 4.1 An outline of the ion mobility cell in the Synapt G2 instrument. This is a schematic view of the TriWave cell where the separation of the ions according to their mobility takes place. The three main regions of the IM cell (trap, IM separation and transfer) and labelled on the diagram. The black lines represent the electrode pairs. Adapted from Pringle et al. (2007).

In the transfer region a travelling wave voltage is continuously applied in order to maintain the mobility separation of the ions as they travel to the TOF and then the detector. A main advantage of this instrument is that it allows the simultaneous collection of both ATD data and mass spectra which is achieved by synchronising the TOF acquisition with the release of ions from the trap SRIG into the IM SRIG (Pringle et al., 2007).

4.2.4 Calibration of TWIM-MS data

In order to obtain a good estimate of the collisional cross section from ATD values for an unknown molecule, the ATD data must be calibrated using the mobilities of standards for which there are established cross sections. Different research groups have developed various approaches to calibrating ATD data. A calibration method has been previously published by the Scrivens group (Thalassinos et al., 2009) and was used throughout this work. The size of the unknown molecule dictates which standard will be used for calibration and for the experiments on HSA

Trap Ions from quadrupole Gate IMS Helium 180 ml/min Nitrogen 90 ml/min Transfer Argon 3 ml/min Ions to TOF

92 reported here, sperm whale myoglobin was used as a calibration

standard. Importantly the calibration standard must be analysed under

the same conditions as the unknown sample (Leary et al., 2009) as the

buffer gas, T-wave height and wave velocity can have an impact on the

ATD of an ion. For the estimation of protein cross sections, a power fit

series has been shown to be the most appropriate (Thalassinos et al., 2009).