Uv6000lp diode array detector

The simultaneous separation and quantification of the free Co-SH and acetyl-CoA in deproteinized brain extracts (20 µL) was performed by high performance liquid chromatography (HPLC) according to methods formerly set up in our laboratory [56 (link),58 (link)] using a Hypersil C-18, 250 × 4.6 mm, 5 µm particle size column, provided with its own guard column (Thermo Fisher Scientific, Rodano, Milan, Italy). The HPLC apparatus consisted of a SpectraSystem P4000 pump system (Thermo Fisher Scientific, Rodano, Milan, Italy) and a highly-sensitive UV6000LP diode array detector (Thermo Fisher Scientific, Rodano, Milan, Italy), equipped with a 5 cm light path flow cell and set between a 200 and 300 nm wavelength. Assignment and calculations of free Co-SH and acetyl-CoA in the chromatographic runs of brain samples were performed by comparing the retention times, absorption spectra, and area of the peaks (calculated at 260 nm wavelength) of the chromatographic runs of mixtures containing known concentrations of true free Co-SH and acetyl-CoA.

The simultaneous separation of high-energy phosphates (ATP, ADP, AMP, GTP), purine nucleosides (inosine, adenosine, guanosine) and oxypurines (hypoxanthine, xanthine, uric acid) in the protein-free cell extracts (200 µL) was carried out using previously established ion pairing HPLC methods which utilize tetrabutylammonium hydroxide as the pairing reagent [63 (link)]. Separation was obtained using a Hypersil C-18, 250 × 4.6 mm, 5 µm particle size column, provided with its own guard column (Thermo Fisher Scientific, Rodano, Milan, Italy). The HPLC apparatus consisted of a SpectraSystem P4000 pump system (ThermoFisher Scientific) and a highly-sensitive UV6000LP diode array detector (ThermoFisher Scientific), equipped with 5 cm light path flow cell and set up between 200 and 300 nm wavelength. Assignment and calculations of the compounds of interest in chromatographic runs of cell extracts were performed at 260 nm wavelength by comparing retention times, absorption spectra, and area of the peaks of chromatographic runs of mixtures containing known concentrations of true ultrapure standard mixtures.