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14 February 2017
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High-Resolution Liquid Chromatography coupled to UV-Vis and MS (HPLC-UV/Vis-MS) is a technique of great importance, as it allows to separate, analyse, quantify and characterise mixtures of little or no volatile products.
High Resolution Mass Spectrometry is a widely used technique for the determination or confirmation of the composition of compounds. Això és degut al fet que s'obté la m/z de l'ió amb un error <5 ppm i, per tant, permet restringir dràsticament les fórmules moleculars assignables. In many cases an elemental and isotopic composition can be assigned to the molecule. In addition, mass-mass spectrometry (MS/MS), i.e. the study of the breaks that an ion undergoes when it is supplied with energy, provides structural and connectivity information of the molecule.
1 mg of undissolved sample (preferably) in a new glass vial is required, accompanied by the sample request showing the molecular formula, structure and reference of the sample. If the sample is delivered dissolved it cannot contain precipitated material, in which case it should be filtered. On the other hand, the solvent must be reported, and if it is a mixture, the components must be reported, if possible.
For column injection, the sample must be completely soluble in the mobile phase and must be filtered. For column injection, the sample must be completely soluble in the mobile phase and must be filtered.
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.
Mass spectrometry is based on the ionisation of the sample (allows the analysis of both positive and negative ions) and the subsequent separation of the ions (molecular ion and fragments) in the gas phase depending on their mass/charge ratio (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, whose intensities are proportional to their respective abundances.
The dissolved sample is introduced into the injection port 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. Buffers can also be used to improve the separation of compounds. Finally, the separate components can be detected, characterised and/or quantified using different types of detectors, such as the mass spectrometer.
Electrospray (ESI): It is one of the mildest ionistions. Ions are generated by applying a potential difference to a spray formed from the sample solution and an inert gas (N2). The solvent in the small charged droplets evaporates and the analyte molecules approach and repel each other, until finally, when the repulsion of charges of the same sign is greater than the surface tension, the droplets explode. This process is repeated until the analyte is free of solvent. Cryospray (Cryo): It allows the generation of ions at low temperature (up to -100°C), avoiding the decomposition of the sample. The nebulisation and drying gas are cooled using cold nitrogen gas.
Atmospheric Pressure Chemical Ionization (APCI): Makes it possible to handle higher workflows and samples with high polarity. First, the eluent from the UHPLC is nebulised and vaporised at other temperatures. Next, it comes into contact with an ionised gas that is responsible for transferring the ionisation to our sample.
Quadrupole Time-of-Flight (QTOF) mass detector: Allows positive or negative ions to be detected. The mass range is from 50 to 20,000 or 40,000 m/z. There are three ion sources, Electrospray (ESI), Atmospheric Pressure Chemical Ionization (APCI) (allows higher flow rates [as it works at higher temperatures] and non-polar solvents; generates more fragmentation), Cryospray (makes it possible to work at a low temperature (up to -100ºC) with direct injection only. On the other hand, the qTOF analyser enables MS2 analyses, that is, the study of breaks that an ion undergoes when provided with energy, which brings information on the molecule's structure and connectivity. In addition to being used as a detector for HPLC, pure samples can be analysed or reactions can be followed by directly infusing the sample using a continuous infusion syringe pump.
Charged ions are separated by their mass/charge ratio (m/z). Ions with n charges are detected at mass/n in mass units. The n charged isotopic peaks are spaced 1/n apart in mass units. This allows easy identification of the charge state using the resolved isotopic spectrum.
The high-resolution hybrid quadrupole/time-of-flight analyser allows us to obtain MS/MS spectra with high resolution on the fragment ions.
For mass spectrometry the resolving power of the quadrupole is turned off. In this case, the quadrupole works as an additional guide. In contrast, for mass-mass spectrometry it is used as a mass filter to isolate a certain ion or a defined mass range. In the collision cell Collision Induced Dissociation (CID) of the selected ions takes place. The isolated "parent" ions are accelerated and collide with a neutral collision gas (N2). In this way, fragments or "daughter" ions are generated, obtaining an MS/MS spectrum that provides information on the structure of the molecule.
In the time-of-flight analysr the ions are accelerated orthogonally and pass through a field-free region where they are separated according to their m/z ratio. This separation is due to the fact that ions with a fixed kinetic energy, if they have different m/z values, are accelerated at different speeds. The determination (m/z) takes place by the precise measurement of the drift time after the orthogonal acceleration of the ions until they hit 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. In addition, the time-of-flight quadrupole analyser allows MS2, that is, the study of the breaks that an ion undergoes when energy is supplied, which provides structural and connectivity information of the molecule.
In addition, the option to add the ionisationcryoespray method allows the identification of species at low temperatures (as low as -100°C) facilitates the detection of thermolabile molecules and reaction intermediates with short lifetimes that could not be detected under normal ESI ionisation conditions (T>150°C).
It is possible to ionise and characterise very precisely molecules with a 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 the capacity to encapsulate fullerenes. The characterised structure is highly complex and has a molecular mass greater than 12000 g/mol. In addition, the technique allows us to monitor the encapsulation of different fullerenes and evaluate the affinity of the box for each of them.Further information: “Sponge-like molecular cage for purification of fullerenes”. Nat. Commun., 2014, 5, 5557.
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.
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).
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.
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