Gx 271 liquid handler

N₂O fluxes between the soil and the atmosphere were measured in the study areas (Table 1) during the summer of 2013 [13 ] by using the static chamber method [62 ]. N₂O flux measurements were done once a month in June, July and August on polyvinyl chloride collars (6 collars per plot; diameter 0.22 m and height 0.05 m). The gas samples taken from chambers (height = 0.24 m and diameter = 0.2 m) were analyzed by an Agilent Gas Chromatograph model 7890A (GC, Agilent Technologies, USA) equipped with an autosampler (Gilson GX-271 Liquid Handler) by using a Flame Ionization Detector. Samples were analyzed using six-point standard curve [62 ]. The N₂O fluxes were calculated by using linear regression fitted to time and concentration change inside the chamber headspace. The annual N₂O fluxes for different aged stands were calculated by converting the measured amount of N₂O (mg m^-2 s^-1) per hectare and multiplying them by the average length of the growing season (120 days) of the study area. The annual N₂O fluxes for non-measured periods were linearly interpolated from sequential measured values.

HPLC-DAD for analysis was performed on Gilson GX-271 Liquid Handler with system 322 H2 Pump connected to a 171 Gilson Diode Array Detector and fraction collector. A XBridge BEH C18 XP Column (130 Å, 2.5 μm, 4.6 mm × 100 mm) with XBridge BEH C18 XP VanGuard Cartridge (130 Å, 2.5 μm, 3.9 mm × 5 mm) was used for analytical run with a flow rate of 0.8 ml/min. HPLC-grade water with 0.5% formic acid (solvent A) and HPLC-grade acetonitrile with 0.5% formic acid (solvent B) were used with the following gradients: 0 min, 20% Solvent B; 2 min, 20% Solvent B; 15 min, 95% Solvent B; 18 min, 95% Solvent B; 20 min, 20% Solvent B. Data acquisition and procession for the HPLC-DAD were controlled by TRILUTION LC V3.0.