Mass Spectrometry Techniques

ESI analyses are generally performed on the LCT Premier QTOF or the Orbitrap Q Exactive Plus using capillary spray. ESI mass spectra pritouts will clearly state at the bottom which instrument and at the top which polarity has been used to acquire the data. For capillary ESI different source ("cone") voltages may be used to either promote precursor fragmentation ("harder" ionisation) or to minimise it and promote multiple charging ("softer" ionisation). Spectra are labelled appropriately when different source voltages give different results. Electrospray data acquired on the Orbitrap Q Exactive Plus to m/z 3,000 are generally also "accurate mass measurement" (5 ppm guaranteed; usually better than 2 ppm).

ESI analyses are generally performed on the Orbitrap Q Exactive Plus using capillary electrospray with a HESI ion source.

HESI analyses are performed on the Orbitrap Q Exactive in positive or negative ionisation mode (one polarity per analysis). The Vanguish LC system is used with a 96-well autosampler plate with a split loop injection. Each sample uses its own sample vial.  Mobile phases and gradient and Orbitrap Q Exactive conditions: see

Method Document for Orbitrap

Method Document for Orbitrap

Loop-injection method by a Waters 2720 autosampler is used at the flow rate 200 µL/min., through a 10-µL sample loop into which the sample is injected. Solvents used water – methanol (50:50) in 0.1 % formic acid. Sample preparation: majority of the samples (1-5 µL) dissolved in 1 mL methanol or acetonitrile or dichloromethane or water.  

Capillary ESI source parameters:- Source Temperature: 80°C; Capillary ESI ionising voltage: positive ionisation: +2.5kV; negative ionisation: -2.0kV; Cone gas N₂: 60L/hr.; Desolvation gas N₂: 100 L/hr.

QTOF set in W-mode and calibrated internally and externally with sulfadimethyhoxydine generating [M]⁺ ions at m/z 311.0814.

ESI sample requirements (wide range of analytes): especially suitable for polar or thermo-labile samples; ESI specialities are salts, non-covalently bound species, multiply charged complexes and very high MW (bio)molecules that will multiply charge.  MALDI rather than ESI is generally used for high mass species.

Accurate mass Electrospray (ESI) is the preferred laboratory method and is attempted for all samples submitted for accurate mass measurements. Other techniques will only be used if ESI fails. Negative and Positive ionisation scans are done on the Orbitrap Q Exactive Plus.

APCI in this facility is performed on the LTQ and on the Orbitrap Q Exactive Plus.

Using the Orbitrap Q Exactive Plus it is possible to perform Atmospheric solids analysis probe (ASAP) analysis. Samples are deposited onto a glass capillary as a solid or in solution and inserted next to APCI probe.  

EI and CI analyses are generally performed on the ThermoScientific GC-MS single quadrupole instrument or MAT900 magnetic sector. The GC-MS is used for volatile samples and mixtures, and the MAT900 is used for less volatile compounds, for metal-containing species, and when there are special sample requirements, e.g. insoluble samples.

EI: Source temperature 200°C; electron energy 70eV; mass range m/z 33-1,300 unless higher is requested.

CI: vary with application - available on request; mass range m/z 33-1,300 unless higher is requested.

Accurate mass EI and CI measurements, in positive ionisation mode, are obtained on the MAT900 by "peak matching", with mass resolution between 8,000 and 10,000 (10% valley definition). For EI, Heptacosa (perfluorotributylamine) is the usual reference compound for accurate mass measurement, and for CI, PEG (polyethyleneglycol) is usually used. If a different reference compound is used, it will be specified in an attachment to the result. Precision of the method is better than 3ppm and nearly always within 1ppm. Usual mass range is 50 to 1,000 Da.

• CI source temperature is 140°C and electron energy 50 eV; the solid probe is used.
• EI electron energy 70 eV; the solid probe is used; source temperature depends on sample.

GC-MS is performed on the Thermo Scientific Trace 1310 GC connected to an ISQ single quadrupole mass spectrometer using EI or CI with ammonia (or methane) in either polarity. The GC column is a TR-5MS, 30m x 0.25mm x 0.25µM column (Thermo Scientific, UK) or DB5 (95% methyl and 5% phenyl). GC conditions depend on those specified in the analysis request. Otherwise, samples will be analysed using GC-MS methods established in the mass spectrometry facility by staff. So please specify the boiling point of compounds. Analysis of liquid or gas samples may be done. More complex applications are available on a collaborative basis with facility such as SPME, headspace injections, liquid injections and various other options (most with automation if high-throughput required).

• Sample introduction: liquid injection; head-space injection; SMPE
• Sample incubator is available temperature from 30 °C to 200 °C
• GC conditions: TR-5MS, 30 m x 0.25 mm x 0.25 µM column
• MS conditions: EI: from 200°C to 280°C; electron energy 70 eV; mass range m/z 33 to 1200.

LC-MS may be performed on the LTQ, Agilent 6510 LC Q-TOF system and the Orbitrap only by special arrangement with the facility, on collaborative basis. Please contact the facility staff for details.

MS/MS may be performed by special arrangement with the facility, usually on a collaborative basis. Please contact the mass spectrometry staff for details.  Different methods of tandem operation are available, in various ionisation modes: Collison induced dissociation (CID), HCD (high energy CID) or PQD (pulsed- dissociation) on Orbitrap and Agilent Q-TOF with accurate mass of fragment ions (ESI). Whereas the LTQ has capability of MSⁿ. Also available data-dependent acquisition on Orbitrap, Agilent Q-TOF and LTQ.

MALDI is performed on a Waters micro Mass MALDI-TOF spectrometer. A typical procedure involves mixing solutions of sample and matrix, and pipetting 3-µL onto the target well of a sample plate.

The sample spot is dried, allowing co-crystallisation of the mixture, then irradiated with a pulsed N₂ laser (200 Hz solid-state, 355nm).

The matrix is ablated from the plate while the sample is simultaneously desorbed and ionised, then accelerated into a flight tube (typically 20 kV). The instrument may be run in any combination of positive or negative and linear or reflector modes.

MALDI matrices in the facility could be found in the fridge: