3.3 Purification of γ-tocomonoenol by column chromatography
Depending on CCC fraction and CCC run, the purity of γ-T1 (7 ) ranged only between 3.3% and 17.8% after CCC separation. This was mostly due to the low share of γ-T1 in the sample. More abundant compounds result in broader peaks which are overlapping with minor compounds such as γ-T1 in the present case (Müller et al., 2019).
Specifically, the presence of high amounts of phytol-like compounds in some CCC fractions reduced the purity of γ-T1. With regard to tocochromanols, γ-T1 was usually predominant (maximum share 93.5%), followed by δ-T and β-T1. Yet, these two tocochromanols show the same ECL as γ-T1 which makes it difficult to separate them by CCC (Vetter et al., 2019). Hence, highly pure γ-T1 could not be obtained by CCC alone but required rather a complementary method with orthogonal separation characteristics. Recently, Müller et al. showed that column chromatography eluted tocochromanols predominantly according to the methylation pattern on the 6-chromanol ring, specifically in the order α- < β- and γ- < δ-tocochromanols (Müller et al., 2020). Our initial tests confirmed this because - contrary to CCC - γ-T (14 ), γ-T1 (7 ) and γ-T3 (1 ) could not be separated by column chromatography. Especially, presence of γ-T (7 ) which showed a very broad elution range in CCC due to its high concentration was a problem in late CCC fractions (Fig. 3 ). Likewise, presence of β-T1 (9 ) was unfavourable, because it could not be separated from γ-T1 by column chromatography. Therefore, only CCC fractions comparably rich in γ-T1 (7 ) but with negligible amounts of β-T1 (9 ) and γ-T (14 ) were considered for the isolation. This prerequisite was fulfilled with CCC fractions with 82-89% CEV in order to reach a purity of >95% γ-T1 (Fig. 3 , dotted lines). Though, this constraint implied that a large share of γ-T1 could not be pooled.
For the majority of CCC runs, CEV range 81-91% was also suitable and in some runs the range 79-92%, additionally. Due to the presence of more abundant phytol- and farnesol-related compounds (section 3.2), the amount of γ-T1 (7 ) was only 0.01-0.51 mg. However, these major compounds still had a strong impact because the capacity of the column was only ~2 mg. Therefore, suitable CCC fractions could not be pooled but had to be chromatographed individually. While hydrocarbons like squalene (elution into fraction 1) (Hammann et al., 2015) could be separated, phytol-like substances and shares of the farnesol-like compounds (parts eluting into silica fraction 4) were also detected into the tocochromanol fraction 3. Therefore, silica fraction 3 was subdivided into six sub-fractions according to Müller et al. (Müller et al., 2018) (section 2.6) with slight modifications. Subsequent GC/MS analysis of the silylated silica fractions showed that γ-T1 (7 ) eluted mainly into silica fraction 3.6 which also contained the majority of impurities (Fig. 7 ). Hence, purity of γ-T1 in fraction 3.6 only was between ~1% (Fig. 7a ) and ~25% (Fig. 7b ). Despite some variations from run to run (Fig. 7 ), the purity of fractions 3.3, 3.4 and 3.5 was usually >95% (Fig. 7 ). Accordingly, this fractionation scheme was applied to all suitable CCC fractions from different CCC runs, and fractions 3.3, 3.4, and 3.5 were measured by GC/MS and pooled if pure enough, while silica fractions 3.2 and 3.6 were combined and chromatographed again. Altogether, ~45 separations on the silica column were carried out, each of which allowed to collect between 0.06 mg to 0.18 mg of highly pure γ-T1 (7 ). Finally, CCC and column chromatography provided 6.8 mg γ-T1 (7 ) with a purity of 96.0%. Minor impurities originated from traces of β-T1, γ-T (Fig. 8 ) and two phytol-like compounds. Hence, the expenditure in the lab was very high (saponification of ~2 L PSO, eleven CCC runs, 45 silica columns, >250 GC/MS runs). However, the goal could be reached and the isolate of γ-T1 (7 ) could be subjected to NMR analysis.