1260 infinity refractive index detector

The methylating efficiency of recombinant MBCOMT was evaluated by measuring the amount of metanephrine using epinephrine as substrate and as previously described [42 (link)]. Briefly, the MBCOMT lysates were incubated at 37°C for 15 min, using epinephrine as substrate and the reaction was stopped with 2 M of perchloric acid. Then, after processing the samples [42 (link)], the metanephrine levels in the samples were determined using HPLC with electrochemical detection in a coulometric mode, as previously described [43 ]. On the other hand, the levels of glycerol and methanol in the culture broth were quantified using a HPLC coupled to a 1260 Infinity Refractive Index Detector (Agilent, Santa Clara, CA, USA), according to what was previously described [21 (link)]. The chromatographic separation was achieved on a cation-exchange analytical column Agilent Hi-Plex H (300 × 7.7 mm i. d.; 8 µm) and the analysis was performed at 65°C with a flow rate of 0.6 mL/min using isocratic elution with 0.005 M H₂SO₄. The samples were centrifuged at 6,000 rpm for 10 min and the supernatant was filtered prior the injection through a 0.22 µm cellulose-acetate filter.

Deacetylated chitosan oligomers were purified on three HiLoad 26/600 Superdex 30 pg columns (GE Healthcare Europe GmbH, Freiburg, Germany) in a row with an overall dimension of 2.60 × 180 cm. Ammonium acetate buffer (0.15 M, pH 4.5) was used as mobile phase and the flow rate was set to 0.8 ml min⁻¹. The effluent was monitored with an online refractive index detector (1260 Infinity Refractive Index Detector, Agilent Technologies Deutschland GmbH, Böblingen, Germany) which was coupled to a datalogger13 (link)37 (link). Fractions containing oligomers were pooled and analysed by UHPLC-ELSD-ESI-MS.