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.