2.4. Dynamic Headspace (DHS) Analysis. Volatile
compounds were extracted from samples of fresh crop material
of three Thymus species using a Master dynamic headspace
(DHS) with purge and trap together with an automatic sampler
(DANI Instruments S.p.A., Milan, Italy). Plant material was
immediately processed after being harvested to ensure that
conditions were the most similar to those of the living plant.
Aerial parts with an average weight of 0.31 ± 0.01 g were placed
on the bottom of 20 mL closed-cap headspace vials and were
directly placed inside the DHS autosampler which has temperature
control. The DHS technique consists of five steps (incubation,
stripping, trap dry step, injection, and baking), and it is the
experimental methodology that currently allows the highest
concentrations of volatiles for chemical analysis. Conditions used
in this work are similar to those of Reboredo-Rodriǵ uez et al.12
with minor changes as summarized ahead.
First, in the incubation step, the vial containing the crop
sample is preheated at 60 °C for 15 min inside the DHS oven
and shaken in fast mode. Then, in the stripping step, the vial is
pierced by the double needle and flushed with a flow of
nitrogen (inert gas) at 150 mL/min for 90 min, keeping the
temperature at 60 °C. The stripped volatile compounds are
then concentrated in a cooled trap (90 mm long quartz tube
with a 4 mm o.d.) filled with Tenax GR 60−80 mesh for
approximately 4 cm. Afterward, in order to remove part of the
humidity from the trap prior to GC injection, the trap dry step
was performed for 1 min at 0 °C. At the end of the dry step the
switching valve rotates, and the trap is disabled from the
auxiliary gas (nitrogen) circuit, so that the carrier gas (He N60)
is passed through the trap and heated at 250 °C, and then
passed through the dew stop device kept at 0 °C to avoid
humidity in the loop. Trap desorption and injection phase run
for 15 min. Finally, in the baking step the system is flushed with
auxiliary gas at a flow rate of 80 mL/min for 15 min in order to
condition the trap (heated at 260 °C) and remove traces of
condensed water or sample analytes.