It is necessary 1 mg of sample without dissolving (preferably), the structure and the reference of the sample, in avenue of new glass, accompanied of the application of sample where the molecular formula is shown. If the sample gives itself up dissolved it cannot contain hasty material, in this case it would be necessary to filter it. On the other hand, it be necessary to inform of the solvent, and if it is about a mixture it be necessary to inform, if possible, of the components of this.

Regarding the injection through column, the sample has to be totally soluble in the mobile phase and has to be filtered. If the use of tamponned mobile phases is necessary the user will have to provide these. 

You will need to give the toxicity and any storage precautions. Once the report has been delivered, if you do not collect the sample within one week, any remaining sample will be destroyed. 

The spectrometry of masses founds in the ionisation of the sample (so much allows to analyse positive ions how negatives) and in the posterior separation of the ions (molecular ion and fragments) in gas phase depending on its relation too much/load (m/z).

L’analitzador de temps de vol (TOF) permet assignar la massa dels ions registrats amb una gran precisió (error<5 ppm), fent ús de la dependència de la velocitat amb la relació massa/carrega.

These ions are detected as ionic currents, the intensities of which are proportional to its respective abundances.

Introduction of the sample through UHPLC

The dissolved sample works in into the port of injection of the chromatograph. The sample passes through the chromatographic column thanks to the bombardment of the mobile phase that is a mixture of solvents. The different interaction of the anàlits with the mobile phase and with the stuffing of the column allows the separation of the components of the mixture. This technique allows to work at high pressures and this the linear speed of the compounds makes increase in the column and reduces the diffusion, improving the speed and the resolution of the chromatography. Can be worked of way isocràtica (with the same one always mix of solvents) or a gradient can be used. Tampons can also be used to improve the separation of the compounds. Finally, the separate components can be detected, characterised and/or quantified through different types of detectors, as for example the spectrometer of masses.

Sources of ionisation

Electroesprai (ESI): It is one of the softest ionisations. The ions are generated through the application of a difference of potential in a spray formed with the dissolution of the sample and an inert gas (N₂). The solvent that there is in the small loaded drops keeps on evaporating and the molecules of anàlit approximate and are repelled, until finally, when the repulsion of the loads of the same sign is bigger than the surface tension, the drops explode. This process is repeated until the anàlit remains free of solvent.

Cryoespray (Cryo): Allow the generation of the ions at low temperature (until -100 °C), avoiding the decomposition of the sample. The gas of nebulisation and of drying they catch a cold using gas nitrogen cold.

Atmospheric Pressure Chemical Ionization (APCI): Allow to work with higher workflows and samples with a high polarity. Firstly, the eluent coming from the UHPLC is nebulitzat and  vaporised at other temperatures. Next, the person in charge is put in touch with an ionised gas that it is of transferring the ionisation to our sample.

Detectors

• Detector of variable wave length (VWD) for analyses of a λ: Register in continuum the length of programmed wave. The source of radiation comes from a lamp of discharge of arch of deuterium (rank of wave lengths: 190-600nms). A mass spectrometer can be connected to the detector's output in series, to extract complementary information.

• Detector of masses Quadrupol Temps of Flight (QTOF): Allow to detect positive or negative ions. The rank of masses is from 50 to 20,000 or 40,000 m/z. Three sources of ions are counted on, Electrospray (ESI),  Atmospheric Pressure Chemical Ionization (APCI) (allow to use higher flows (since it works at higher temperatures) and non polar solvents; generate more fragmentation), Criosprai (allow to work temperature to casualty (until -100ºC) only with direct injection.
  On the other hand, the analyser qTOF allows to analyse MS², that is, the study of the breakings that an ion suffers when energy provides it, what brings information structural and of connectivity of the molecule.
  Besides being used as a detector for the HPLC, pure samples can be analysed or reactions introducing the sample in a direct way for infusion through a bomb of infusion syringe can be followed continue.

Analysers

The loaded ions are sorted out by its relation too much/load (m/z). The ions with n loads are detected in too much/n in units of too much. The isotopic pikes n loaded they find long-distance 1/n in units of too much. This allows an easy identification of the state of load through the solved isotopic spectre.

The hybrid analyser quadrupol/temps of flight of high resolution allows to obtain spectres MS/MS with high resolution in the fragment ions.

For the spectrometry of masses the power of resolution of the quadrupol goes out. In this case, the quadrupol works as an additional guide. However, it is used by the spectrometry of masses-masses as a filter of masses to isolate a certain ion or a rank of masses defined. In the cell of collision the Dissociation Induced for Collision (CID) is produced del/s selected ions. The isolated ions “parents” speed up and collide with a neuter gas of collision (N₂). In this way the fragments or ions “children” obtaining a spectre from MS/MS that gives information about the structure of the molecule are generated.

In the analyser of flight time the ions are speeded in an orthogonal way and go through a region free of fields in the one that they are separate according to its relation m/z. This separation is due to the fact that the ions with a kinetic energy fixed, if they have different values of m/z, are speeded at different speeds. The determination (m/z) takes place after the orthogonal acceleration of the ions for the precise measure of the time of drift until strikes in the detector.

There are numerous applications for this technique, as it allow separations, characterisations and quantifications of substances that are hardly or not-at-all volatile, of diverse nature and provenance.  The possibility to use several types of chromatographic columns together with  the option to select different detectors(some of them can be used successively), they make that the extraction of a big volume of information is possible while allowing to approach a very  extensive range of needs analytics.

Specially useful the use of the detector of masses is since I allow to characterise pure molecules or characterising and quantifying you mix of products when it is coupled to HPLC. Besides, the analyser quadrupol time of flight allow to make MS², that is, the study of the breakings that an ion suffers when energy is provided to him, which brings information structural and of connectivity of the molecule.

Besides, the option to couple the method ofionisation cryoespray allow the identification of species at low temperature (until -100 °C) and, therefore, facilitate the detection of thermolabile molecules and ofintervals of reaction with short time of life that in the normal conditions of ionisation for ESI (T>150 °C) they could not be detected.

Characterisation of organic molecules and complexes with transition metals with ESI-MS

It is possible to ionise and to characterise molecules with great accuracy with very diverse and complex structure.

Exemple concret: A diferència dels espectròmetres de masses de baixa resolució (generalment l'error en la massa d'aquests aparells és de ±0,2 unitats) el QTOF obté una gran exactitud en la m/z que permet distingir entre molècules amb m/z molt similar (error<5 ppm, per una molècula de m/z=300 seria ±0,0015).

Concrete example: Characterisation of supramolecular boxes with capacity to encapsulate ful·lerens. L'characterised structure is of great complexity and have a molecular mass superior to 12000 g/mole. Besides, the technique allows us to monitor the encapsulation of different ful·lerens and to evaluate the affinity of the box for each of them. More information: “Sponge-like molecular cage for purification of fullerenes”. Nat. Commun., 2014, 5, 5557.

Characterisation of thermolabile molecules using cryospray

Nebulisation and drying gas temperatures can reach -100ºC, while temperatures between 150ºC and 250ºC are normally used, which usually causes the decomposition of these types of molecules. Practical example: The reaction of 1 in acetone at -90 °C with oxygen causes the formation of a type of Cu (III) with a bis-oxo bridge. When adding 3 equivalents of sodium 2,6-di-fluoro-benzoate a purple type is obtained that corresponds in the coordination of the latter to the type of Cu(III). These species are highly reactive and have very short life spans even at -90 °C. However, they have been observed in our laboratory through the use of the cryospray. Further information: Selective Ortho-Hydroxylation–Defluorination of 2-Fluorophenolates with a Bis(μ-oxo)dicopper(III) Species. Angew. Chem. Int. Ed., 2014, 53(36), 9608–9612.

Obtaining structural information through MS-MS

The ions obtained when measuring a sample (even those obtained in cryospray conditions at -90ºC) can be isolated and made to collide in a controlled way for the purposes of obtaining the structural information on the molecule through the analysis of the fragments obtained after collision. For example, in the above case, having isolated and applied some energy, the peak of the complex with phenolate loses it on colliding with the collision cell’s N₂ molecules. This allows us to say that this compound is found in the molecule without reacting and that we are therefore observing the ion of coordination and not of the reaction of this molecule with the Cu (III).

• Pamphlet and specifications of the Bruker Elute
• Pamphlet and specifications of the Bruker Compact
• Pamphlet and specifications of the Bruker TargetScreener
• Pamphlet and specifications for the Bruker micrOTOF-QII

The acquisition of this equipment was subsidised by Ministerio de Ciencia e Innovación through the INNPLANTA INP-2011-0059-PCT-420000-ACT1 project and co-funded by the European Regional Development Fund (FEDER) and the Starting Grant ERC-2011-StG-277801 European project.