2.6 Column chromatography
CCC fractions with highest purities of γ-T1 according to GC/MS analysis
(generally CCC fractions 16-19, partly also CCC fractions 15 and 20 and
scarcely CCC fractions 14 and 21) were further purified by column
chromatography (1 cm inner diameter glass column filled with 5 g silica
gel 60, deactivated with 20% water) according to Hammann et al. (2015).
The selection criterion was a maximum of 5% interfering
β-/γ-tocochromanols in relation to γ-T1 in the fraction. Selected CCC
fractions were transferred to the column using a Pasteur pipette. For
this purpose, the complete solution from the 2 mL brown glass GC vial
(section 2.4) was individually placed onto the column and the vial was
rinsed three more times with 0.5 mL of n -hexane. Silica fraction
1 (30 mL n -hexane) and silica fraction 2 (40 mLn -hexane/ethyl acetate, 99:1, v/v ) were collected in 100
mL pear shaped flasks. Based on initial trials, the subsequent silica
fraction 3 (n -hexane/ethyl acetate, 95:5, v/v ) was
subdivided into six aliquots of different volume, namely fraction 3.1
(0-15 mL), fraction 3.2 (15-17 mL), fraction 3.3 (17-20 mL), fraction
3.4 (20-22 mL), fraction 3.5 (22-25 mL) and fraction 3.6 (25-50 mL).
Fractions 3.1 to 3.5 were collected in 20 mL brown glass derivatisation
tubes and fraction 3.6 in a 100 mL brown glass pear shaped flask.
Finally, more polar compounds were eluted with 40 mL ethyl acetate
(silica fraction 4) into a 100 mL pear shaped flask. Fractions 1, 2 and
4 were evaporated to dryness in a rotary evaporator and fractions
3.1-3.6 by means of a gentle stream of nitrogen. Residues of all
fractions were taken up with n -hexane and transferred into 2 mL
brown glass GC vials, respectively. Aliquots were trimethylsilylated and
measured by GC/MS (section 2.5).