RESUMEN
We investigated how physico-chemical properties of charged droplets are affected by the electrospray process, using simultaneous in situ measurements by laser-induced fluorescence (LIF), Fraunhofer diffraction and mass spectrometry. For this purpose, we implemented a laser-induced-fluorescence profiling setup in conjunction with a fast, high-resolution particle sizing scheme on a modified Agilent Jet Stream electrospray source coupled to a single quadrupole mass analyser. The optical setup permits us to profile the solvent fractionation and the size of the droplets as they evaporate in an electrospray plume by measuring both the angular scattering pattern and emission spectra of a solvatochromic fluorescent dye. Mass spectra are recorded simultaneously. These mass spectrometry and optical spectroscopy investigations allow us to study the relation between the observed charge-state distributions of protein anions and physico-chemical properties of evaporating droplets in the spray plume. By mixing water with methanol, a refolding of cytochrome C is observed as the water percentage increases in the plume due to the preponderant evaporation of volatile methanol.
Asunto(s)
Colorantes Fluorescentes/química , Rayos Láser , Proteínas/química , Citocromos c/química , Espectrometría de Masas , Metanol/química , Tamaño de la Partícula , Solventes/química , Espectrometría de Masa por Ionización de Electrospray , Compuestos Orgánicos Volátiles/química , Agua/químicaRESUMEN
Current nano-LC/MS systems require the use of an enrichment column, a separation column, a nanospray tip, and the fittings needed to connect these parts together. In this paper, we present a microfabricated approach to nano-LC, which integrates these components on a single LC chip, eliminating the need for conventional LC connections. The chip was fabricated by laminating polyimide films with laser-ablated channels, ports, and frit structures. The enrichment and separation columns were packed using conventional reversed-phase chromatography particles. A face-seal rotary valve provided a means for switching between sample loading and separation configurations with minimum dead and delay volumes while allowing high-pressure operation. The LC chip and valve assembly were mounted within a custom electrospray source on an ion-trap mass spectrometer. The overall system performance was demonstrated through reversed-phase gradient separations of tryptic protein digests at flow rates between 100 and 400 nL/min. Microfluidic integration of the nano-LC components enabled separations with subfemtomole detection sensitivity, minimal carryover, and robust and stable electrospray throughout the LC solvent gradient.