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preparative separation of fats and products of their conversion. Ultimately, such automated chromatographic
system should have potential to be used in fats’ standards production.
When transferring separation conditions used in the thin layer chromatography (TLC) to high
performance liquid chromatography (HPLC), it should be noted that in the case of liquid chromatography,
including exclusion chromatography (GPC/SEC), the solvent of the sample should be the same as mobile
phase used. In case of a gradient elution – the solvent should have identical composition as the initial mobile
phase used in elution program. During “classical” size exclusion chromatography, the mobile phase has to
ensure minimization, or preferably, absence of sorption interactions between all components of the sample
being separated and the surface of the pores of the stationary phase. Then the elution of substances form
the column takes place in the order from the largest to the smallest particles. The biggest ones have, as the
“diffusion path”, only a small space of the largest pores of stationary phase at their disposal. Th e smallest
ones have the biggest space – the space of all pores (the largest, medium and smallest). The same applies
to the "classical" size exclusion chromatography, when the sorption interactions between components of
separated sample and the surface of the micro-pores of the column packing are absent. However, in the
case where the mobile phase composition and the stationary phase of the column have been selected in
such a way that, in addition to exclusion effects, weak adsorption interactions take plac e – preferably, the
stronger the lower the molecular weight of the separated sample is – an increase of resolution in such
'designed' separation system should be obtained, as it was observed during the studies carried out by means
of TLC and described in this paper.
Such conditions should take place when the sorption area of stationary phase has a defined pore size,
which is properly and adequately selected for the size of separated particles. At the same time, the
stationary phase should be slightly polar, while the mobile phase is not sufficiently polar to provide complete
elimination of adsorption. When tri -, di- and monoalyloglycerols are subjected to separation in described
conditions, the order of elution seems to be obvious: 1 – TAG, 2 – DAG, 3 – MAG, according to increasing
polarity. However, as it was proved by TLC studies, when the examined sample contained also free fatty
acids (FFA) the elution order was not so clear and obvious. However, FFA are relatively smaller particles
than TAG/DAG/MAG and in case of „classical” GPC/SEC they should be eluted as the last ones, they are
significantly less polar than monoacylglycerols (MAG). The elution order in GPC -NP conditions may depend
on the polarity of the mobile phase and the polarity of the stationary phase. Despite of quite low efficiency of
the column, i.e. a limited number of theoretical plates, there is a possibility to select a stationary phase type
and a type of mobile phase in such a way to obtain good group separation and reversed elution order o f the
last two groups, i.e. MAG/FFA. Studies carried out by HPLC technique, the results of which will be the
subject of another paper, revealed that it is also possible to obtain group separation in relation to polarity,
especially within the FFA and MAG groups. However, the GPC/SEC-NP HPLC columns should than have a
very high efficiency (a very high number of theoretical plates (N)). Therefore, the GPC/SEC-NP conditions
are useful for the initial group separation. In contrast, “detailed” separation, in rel ation to polarity differences,
is advisable during classical NP-HPLC conditions with gradient elution. In the case of highly complex
mixtures separation, it is possible or sometimes indispensable, to firstly perform “initial” separation by HPLC -
GPC/SEC-NP, and then separate respective fractions by means of NP -HPLC with gradient elution
[A. Stołyhwo, unpublished results of studies]. It is particularly advisable when the aim of the separation is to
obtain pure chemical compounds or groups of compounds in preparative scale.
Opposite situation will take place when during the particle size decrease (and the molecular mass
decrease) the hydrophobicity of separated compounds (or group of compounds) will increase and their
polarity will decrease. Such situation occurs infrequently, however is possible. Then, it may be profitable to
link up the GPC/SEC conditions with the conditions of weak hydrophobic interactions GPC/SEC-RP.
Finally, it should be noticed that recent studies [18 -21] showed that the separation of individual
acyloglycerol isomers is possible by means of HPLC and C18 (or similar) stationary phases, with gradient
elution, and with anhydrous mobile phases such as dichloroethane, dichloromethane, chloroform,
acetonitrile, etc. Simultaneously, the columns of “RP-type” should be characterized by a very high efficiency
and high selectivity.
Studies concerning usage of high performance multi -diemnsional (nD) and multicolumn (NC) liquid
chromatography for separation of fats and products of their conversion in semi-preparative scale, in order to
obtain pure chemical compounds or groups of compounds, are the subject of the next research paper.
Podziękowania
(Acknowledgements)
Prace były finansowane przez Narodowe Centrum Nauki numer rejestracyjny projektu badawczego:
N N312 417237, numer umowy: 4172/B/P01/2009/37, pt. "Wykorzystanie chromatografii wykluczania
i adsorpcji do rozdzielania i oznaczania grupowego lipidów i produktów konwersji w tłuszczach jadalnych"
Vol. 7, No 1/2015
Camera Separatoria