2.4 Countercurrent chromatographic (CCC) separation
CCC separations were performed with a Quickprep MK8 system (AECS,
Downend, UK) using the periphery described in details (Englert et al.,
2015). Separations were performed using coil 2 in bobbin 1 and coil 3 in
bobbin 2 of the CCC system (total volume 236 mL) (Müller et al, 2018).
The solvent system n -hexane/ACN/BTF (10:6,5:3,5, v/v/v )
(Müller et al, 2018) was prepared in a 2 L separatory funnel. After
equilibration for about 1 h, the lower phase (LP) and the upper phase
(UP) were separated and individually degassed in an ultrasonic bath. CCC
separation was performed in head-to-tail mode (LP used as mobile phase)
which elutes compounds with decreasing polarity (non-polar compounds
eluting last). Accordingly, the two coils were entirely filled with the
(upper) stationary phase, and rotation was started (870 rpm, maximum
speed). External cooling was used to maintain the temperature of the CCC
centrifuge at 17 °C. Then, (lower) mobile phase was pumped through the
system at 4 mL/min and the effluent was collected in a measuring
cylinder until its breakthrough. The volume was noted, and retention of
stationary phase Sf was calculated to be 67-72%,
respectively. Afterwards, between 0.8 and 1.0 g of saponified PSO
extract (section 2.3) was taken up in a mixture of 4.5 mL of both LP and
UP and the whole sample was injected via a 10 mL sample loop into the
CCC system. After a delay of 50 min, 55 4 mL-fractions were collected in
the fraction collector (total run time 105 min). CCC/UV chromatograms
were monitored at 290 nm. Collected fractions were carefully evaporated
to dryness (evaporator, gentle stream of nitrogen), transferred into 2
mL brown glass GC vials (three times flushed with 0.5 mLn -hexane), evaporated again to dryness and weighed. The residue
was taken up in 1 mL n -hexane. Aliquots of each CCC fraction
(equating a final concentration in the measuring solution of
~100 μg/mL) were trimethylsilylated according to Müller
et al. (2014) and analyzed by GC/MS (section 2.5). In total, 11 CCC runs
were performed to yield the final amount of 6.8 mg γ-T1.
2.5 GC/MS analysis of silylated samples
Tocochromanols and interfering compounds in sample solutions and CCC
fractions were analyzed with a 6890/5973N GC/MS system (Agilent
Technologies, Santa Clara, CA, USA), equipped with a 30 m x 0.25 mm
internal diameter capillary column coated with 0.25 μm 5% diphenyl,
95% dimethyl polysiloxane (Optima 5 HT, Macherey-Nagel, Düren,
Germany). The oven program started for 1 min at 55 °C. Then, the
temperature was ramped at 20 °C/min to 255 °C, directly at 1.5 °C/min to
283 °C and finally at 15 °C/min to 300 °C which was held for 9 min
(total run time 39.8 min). An MPS 2 autosampler (Gerstel, Mülheim,
Germany) was used for sample injection (1 μL) in splitless mode.
Injector temperature was set to 250 °C. The carrier gas helium (5.0
quality, Westfalen company, Münster, Germany) was transported with a
constant flow of 1 mL/min. MS data was collected in full scan mode
(m/z 50 to 650) after a solvent delay of either 7.0 min (method
A) or 15.0 min (method B). Transfer line, ion source, and quadrupole
operated at 280 °C, 230 °C and 150 °C, respectively.