EPSRC National Mass Spectrometry Service Centre

EPSRC National Mass Spectrometry Service Centre at Swansea University.

Instrument Operating Conditions.

CHARACTERISATION SPECTRA: APCI CI EI ESI LSIMS (FAB) MALDI

ACCURATE MASS MEASUREMENTS: APCI CI EI ESI LSIMS (FAB)

MIXTURE SEPARATION AND SPECIAL TECHNIQUES: GC/MS LC/MS MS/MS

Electron Ionisation and Chemical Ionisation (EI and CI)
EI and CI analyses are generally performed on the DSQ-Trace GC/MS quadrupole instrument or MAT95 magnetic sector. The DSQ-Trace GC/MS is used for volatile samples and mixtures, and the MAT95 is used for less volatile compounds, for metal-containing species, and when there are special sample requirements, e.g. air sensitive or insoluble samples.

DSQ-Trace conditions:

EI: Source temperature 200°C; electron energy 70eV; mass range m/z 33-650 unless higher is requested.
CI: vary with application - available on request; mass range m/z 33-650 unless higher is requested.

MAT95 conditions:
Mass resolution is set to at least 400 greater than the maximum mass (10% valley definition);
Solids probe or desorption probe may be used, with manually controlled heating rates.

EI: source temperature 120°C to 200°C; electron energy 70eV; mass range as required by analyte;
CI+ or CI-: source temperature 120°C to 160°C; mass range as required by analyte; ammonia or methane reagent gas.

Accurate mass Electron Ionisation (EI) and Chemical Ionisation (CI) measurements, in positive ionisation mode, are obtained on the MAT95 by "peak matching", with mass resolution between 8000 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 5ppm and nearly always within 3ppm. Usual mass range is 50 to 1000Da but higher is possible; measurements of molecular masses over 1000Da are usually discouraged as being a meaningless exercise.

EI electron energy 70eV; either the water-cooled or desorption probe is used, as required; source temperature depends on sample.
CI source temperature ca. 140°C and electron energy 50eV; either the water-cooled or desorption probe is used, as required.

Fast Atom Bombardment / Liquid Secondary Ion Mass Spectrometry (FAB / LSIMS)

"FAB" (actually LSIMS) analyses, in positive or negative ionisation mode, up to mass ca. 3000 Daltons, are performed on the MAT95.
Caesium ion bombardment is used at 18-22 kV energy on to the sample dissolved in a matrix liquid, usually 3-nitrobenzyl alcohol (NOBA). Resolution is sample dependant, and at least 400 greater than the maximum mass (10% valley definition). FAB is undesirable for compounds of molecular weight less than 250Da, as the matrix ions dominate this region.

Note re "FAB" (LSIMS): this service continues to be offered but is being replaced by other ionisation modes as far as possible.

Accurate mass "FAB" (LSIMS) is performed on the MAT95 in positive and negative ionisation mode. Measurements are performed by "peak matching" at resolution 8000 (10% valley definition). Precision of the method is better than 5ppm. Mass range m/z 150 to 1400, although measurements of molecular masses over 1000Da are usually discouraged as being a meaningless exercise. Glycerol/CsI is the usual reference compound. If a different reference compound is used, it will be specified in an attachment to the result.

Electrospray Ionisation (ESI)

ESI analyses are generally performed on the ZQ4000 or the Orbitrap using nanospray. Printouts will clearly state on the top whether nanospray or pneumatically-assisted electrospray has been used (see both methods below). The spectra achieved by both methods are similar; only the inlet and rate of sample consumption differ. For both spray rates, different source ("cone") voltages may be used to either promote molecular 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 Orbtirap to m/z 2000 are generally also "accurate mass measurements" (5 ppm guaranteed; usually better than 3ppm).

Nanospray: analyses are performed on the ZQ4000 or Orbitrap in positive or negative ionisation mode (one polarity per analysis). The Advion NanoMate inlet is used with a 96-well plate, corresponding transfer tips and 400-nozzle spray-chip. (With this inlet, each sample uses its own well, sample tip and nozzle, effectively eliminating the "memory" problem that is common with standard electrospray.) ZQ acquisition is in "MCA" mode using different "cone" voltages, applied consecutively, each for 30 seconds. Solvents and additives used are listed on the results.

ZQ4000 conditions:
Source Temp.: 45°C; Cone gas nitrogen at 30L/hr. (no desolvation gas);
Nanomate: spray voltage: (+ or -) 1.55kV ± 0.1kV; spray time ca. 2 minutes.

Orbitrap conditions: see SOP document.

Electrospray: analyses are performed on the ZQ4000 or the Orbitrap in positive or negative ionisation mode. Samples are loop injected into or infused in a stream of water / methanol (1:1 at 50µL/min.) using the customer-specified solvent for dissolution. Nebulisation is pneumatically assisted by a flow of nitrogen through a sheath around the capillary, a process sometimes referred to as "Ion spray". On the ZQ4000, an Agilent 1100 LC system is used for sample loop injection and delivery. See Orbitrap SOP for more information on this instrument.

ZQ4000 conditions:
Source Temp.: ca. 100°C; Capillary (ionising) voltage: positive ionisation: +3.5kV; negative ionisation: -3.0kV;
Cone gas N2: 60L/hr.; Desolvation gas N2: 100L/hr.

Orbitrap conditions: see SOP document.

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. (In this laboratory, 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 compounds submitted for accurate mass. Other techniques will only be used if ESI fails. If both a full-scan characterisation and accurate mass are required, both are done simultaneously on the Orbitrap. If only a single ion accurate mass measurement is required, it may be performed on the Orbitrap or on the MAT95 in positive or negative ionisation mode using "peak matching".

Orbitrap conditions: see SOP document. Standard operation uses nanospray on the Advion TriVersa NanoMate.

MAT95: Samples are loop injected into a stream of water / methanol (1:1). Nebulisation is pneumatically assisted by a flow of nitrogen through a sheath around the capillary, a process sometimes referred to as "Ion spray". Accurate mass measurements may be performed by "peak matching" or by electric scanning at resolution 8000 to 10000 (10% valley definition). Precision of the method is better than 5ppm and usually within 3ppm. Mass range m/z 150 to 1400, although measurements of molecular masses over 1000Da are usually discouraged as being a meaningless exercise. Different reference compounds are used depending on the mass range and polarity, and are specified in an attachment to the results.

Accelerating voltage: 5kV; Capillary temp.: 200°C ± 30°C; Spray voltage: positive ionisation: +3.0kV; Negative ionisation: -2.8kV.

Atmospheric Pressure Ionisation (APCI)
APCI in this laboratory is generally performed on the Orbitrap using the Atmospheric Solids Analysis Probe (ASAP) to insert samples into the APCI source. (Traditional flow injection APCI is also available but is rarely required). Samples are deposited onto a glass capillary as a solid or in solution. Additives such as ammonium acetate may also be used on the probe or in the ion source, to assist ionisation.

ASAP conditions:
Source Temp.: sample dependent. If the vapourizing temperature is unknown, the sample is introduced at ambient temperature (circa 40ºC) then the temperature is increased until the sample vaporizes.
Corona Discharge Current: 5µA.

Accurate Mass APCI is also carried out using ASAP under the above conditions. For instrument calibration, please see Orbitrap SOP document (instrument is calibrated using the Advion TriVersa NanoMate). Accurate mass is obtained on all ions in the spectrum simultaneously.

Matrix-Assisted Laser Desorption/Ionisation (MALDI)
MALDI is performed on a Voyager DE-STR spectrometer. A typical procedure involves mixing solutions of sample and matrix, and pipetting 0.5-1 microlitre onto the target well of a sample plate. Inorganic salts are sometimes added to promote ionization. The sample spot is dried, allowing co-crystallisation of the mixture, then irradiated with a pulsed N₂ laser ( 337nm, f = 3 or 20Hz). Solvent-free preparations can also be used. The matrix is ablated from the plate while the sample is simultaneously desorbed and ionised, then accelerated into a flight tube (typically 20kV). The instrument may be run in any combination of positive or negative and linear or reflector modes. Specific details regarding conditions will accompany results.

MALDI Matrices
Using the service - more information about services available for different analytes (including polymer details).
Biochemicals - recommended sample clean-up procedures.

Coupled Gas Chromatography - Mass Spectrometry (GC-MS)
GC-MS is usually performed on the Thermofisher Trace-DSQ-II (or the Agilent 5975C GC/MS), using EI or CI, with ammonia (or methane), in either polarity. Accurate mass GC-MS can also be arranged on a collaborative basis, using the MAT95. The stationary phase of preference is DB5 (95% methyl and 5% phenyl). GC conditions depend on those specified in the analysis request. Analysis of liquid or gas samples may be done. (More complex applications are available on a collaborative basis, with facility available for thermal desorption, SPME, headspace, and various other options, most with automation if high throughput is required.)

GC conditions: 5975: split/splitless or PTV injector - conditions vary with application and are available on request.
GC conditions: DSQ: PTV injector, column: WCOT TR5MS 25m x 0.25mm with 0.25μm film.
MS conditions: EI: Source temperature 200°C; electron energy 70eV; mass range m/z 33-650 unless higher is requested.
MS conditions: CI: vary with application - available on request; mass range m/z 33-650 unless higher is requested.

Coupled Liquid Chromatography - Mass Spectrometry (LC-MS)
LC-MS may be performed on the Orbitrap by special arrangement with the Centre, usually on a collaborative basis. Please contact the Service Manager for details.

Tandem mass spectrometry (MS-MS)
MS-MS may be performed by special arrangement with the Centre, usually on a collaborative basis. Please contact the Service Manager for details. Different methods of tandem operation are available, in various ionisation modes:

Collision induced dissociation (CID), HCD* (high energy CID) or PQD** (pulsed-Q dissociation) on Orbitrap, with low resolution selection of precursor ion and accurate mass of fragment ions (ESI or APCI);
Collision induced dissociation using the MAT95 with "linked scanning" (EI, CI, ESI or LSIMS).

*HCD: ref.: Nature Methods - 4, 709 - 712 (2007); Published online: 26 August 2007; doi:10.1038/nmeth1060 (http://www.nature.com/nmeth/journal/v4/n9/full/nmeth1060.html)

**PQD: PQD, a new patented fragmentation mechanism, is now available on Thermo's 2D linear ion traps, eliminating the "1/3 Rule" or “Low Mass Cut-Off” for MS/MS data. Use of PQD allows for trapping and analysis of structure-informative ions such as b-1, y-1 and immonium ions. PQD also enables the detection of MS/MS reporter ions from isobaric peptide tags such as iTRAQ.

Page last updated: 12^th January 2012