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Serveis Tècnics de Recerca

Cromatografia Líquida acoblada a Espectrometria de Masses

HPLC-MS

La Cromatografia Líquida d’Alta Resolució acoblada a UV-Vis i MS (HPLC-UV/Vis-MS) és una tècnica de gran importància ja que permet separar, analitzar, quantificar i caracteritzar mescles de productes poc o gens volàtils

Sol·licitud de treball

Característiques de la mostra:

Injecció directe a l'espectròmetre de masses: Es necessari 1 mg de mostra sense dissoldre (preferentment) en vial de vidre nou, acompanyat de la sol·licitud de mostra on s’hi mostri la formula molecular, l’estructura i la referencia de la mostra. Si la mostra s'entrega dissolta no pot contenir material precipitat, en aquest cas caldria filtrar-la. Per altra banda, cal informar del dissolvent, i si es tracta d'un barreja cal informar, si és possible, dels components d'aquesta.

Pel que fa a la injecció per mitjà de columna, la mostra ha de ser totalment soluble en la fase mòbil i ha d'estar filtrada. Si és necessari l'ús de fases mòbils tamponades aquestes les haurà de proporcionar l'usuari. 

Cal informar de la toxicitat, precaucions d’emmagatzematge i un cop entregat l’informe si no es ve a buscar la mostra al cap d’una setmana, després d’aquest termini el restant d’aquesta serà destruïda.  

Imprès de sol·licitud de treball:
  Versió en Català English version
Direct Injection MS PDF word PDF word

Equipment’s features

Agilent Technologies LC 1200 you would be (HPLC) coupled to Bruker Esquire 6000 Ion Trap (MS)

Year:
2008
Detector UV-screw:
Detector of variable wave length G1314B
Detector of masses:
Ionic trap Bruker Esquire 6000 Ion Trap
Sources of ionisation: 
ESI and APCI
Accuracy in Masses:
± 0.2 u
Fashions of operation:
Rank of Masses (m/z) Resolution FWHM (one) Speed of scan (u/s) 
  Normal 0.6 13000
Standard 50-3000 High 0.45 5500
  Maxim 0.3 1659
Extesa 200-6000 Normal 5 27000


The HPLC-MS consists of the following modules

  • Vaccum deaerator (Degasser G1322A): Flow speed: up to 10 mL/min. Internal volume: 12 mL for canal.
  • Quaternary pump (Quaternary Pump G1311A): Flow range: 0.001-10 mL/min, allows analyses with gradient (columns with ID: 3-9.4 mm).
  • Automatic sampler for 100 x 1.8 mL vials (ALS G1329A):  Injection range: 0.1-100 μL.
  • Column compartment with thermostat (TCC G1316A): Temperature range: from 10ºC below room temperature to 80ºC.
  • Detector UV-screw.
  • Detector of masses.

How does it work?

The sample dissolved in the mobile phase 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 in order to improve the separation of the compounds. Finally, the separate components can be detected, and/or characterised and/or quantified through different types of detectors.

Sample introduction

The UEM has automatic injector with capacity of until 100 avenues (avenues of 1.8 mL). It be necessary to dissolve the samples in solvents of purity HPLC or superior and microfiltrar-les before being sampled. Amount of sample injected between 0.1 and 100 μL.

Chromatographic separation

The compartment of the column has a thermostat that can keep the temperature constant: of 10 bachelor's degrees under the room temperature until 80°C (columns of until 30 cm). 

Detectors

The equipment has mass and UV-Vis detectors:

  • Variable wavelength detector (VWD) for analyses of a λ: Records in continuum the programmed wavelength of the column's eluent. 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.
  • Mass detector with Ionic Trap analyser (Bruker Esquire 6000): Allows the detection of positive and/or negative ions originating from the column’s eluent. The masses range is from 50 to 3000 m/z and the accuracy ±0.2 m/z. The mass spectrometer works by ionising molecules through a source of ions, and by selecting and identifying ions in accordance with the mass/charge relation (m/z).
    • This equipment has two sources of ions, Electrospray (ESI) Source (softer) and Atmospheric Pressure Chemical Ionization (APCI) Source (allows the use of higher flows (since it works at higher temperatures) and non-polar solvents; it generates more fragmentation).
      On the other hand, the ionic trap analyser enables MSn 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.
      Besides being used as a detector for the HPLC, pure samples can be analysed or reactions followed by introducing samples directly for infusion through a continuous-infusion syringe pump.

Practical cases

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 ionic trap allow to analyse simultaneously the positive mode and the negative, this is, to make the study of molecules that when ionising they form positive or negative ions at the same time. On the other hand, also allow to make MSn, 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.

Case studies:

Determination of enantiomeric purity

Specific example: Determination of enantiomeric separation achieved through the use of MOFs especially designed to enantiomerically enrich racemic products. Full information: "Engineering Homochiral Metal–Organic Frameworks by Spatially Separating 1D Chiral Metal–Peptide Ladders:Tuning the Pore Size for Enantioselective Adsorption", Chem. Eur. J. 2015, 21, 9964–9969.  

Quantification of products and mecanístics studies through isotopic labeling

In this case, besides quantifying the products obtained in a reaction, the HPLC-MS has been used to study the isotopic composition of the products obtained. That way, where isotopically marked reagents have been used, we will be able to find out whether these have ended up being part of the product of the reaction and to what extent. The products marked isotopically are those that contain atoms with an isotopic distribution different to the one that it finds to the nature. For example, the products they contain are 18Or, since the most abundant isotope of oxygen is 16O. Therefore, the presence of18O with higher levels than 0.2% implies participation in the reaction of the isotopically marked products introduced. Isotopic marking studies are a very important source of mechanistic information. Further information: Angew. Chem. Int. Ed.2014, 53(36), 9608–9612.

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

Mass spectrometers can be used alone for characterising organic molecules as well as transition metal complexes. Specific example: Characterisation of the molecule [LFeO(CF3SOUND3)]+  through ESI-MS (m/z= 546.1) and confirmation of the presence of the group Fe=O by monitoring the exchange of the oxo group with H218O. Further information from: "Triggering the Generation of an Iron(IV)-Oxo Compound and Its Reactivity toward Sulfides by RuII Photocatalysis". J. Am. Chem. I am., 2014, 136, 4624-4633.

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