Mmushi T MSc (Microbiology).pdf

The sub-fractions of 90:10 and 85:10 were further re-eluted in a silica gel column using
3:1 DCM:ACE to reduce the impurities in the fraction.
2.5.4.1. Preparative TLC
Both fractions were dissolved in small amounts (c. 2 ml) of acetone in which they would
dissolve and applied in a band across the preparative TLC plates (Silica gel 60 F 254 ) .
The plates for the first fraction was developed with 100 % hexane and the second
fraction with 3:1 DCM:ACE. The bands were visualized under ultraviolet light (254 and
360 nm) before a small part on the side of the plates were sprayed with vanillin-sulphiric
acid and heated with a heating gun. The rest of the bands/compounds on the plates
were protected with glass and an aluminium foil against the damage by spray and heat.
The visualised bands on the side were used as the reference line for scraping the
remaining compound/band on the plate with a grass rod. The components were
collected into separate beakers and crushed into fine powders using a glass rod. The
silica powder was eluted with hexane and acetone for compound A and B, respectively.
The volume of solvent was dependent on the quantity and pigment recovered by
filtration through a cut glass pipette plugged with cotton wool to facilitate the removal of
impurities. The process was repeated thrice or until the silica gel powder regained its
original colour. Each purified sample was then evaporated and weighed and thereafter
transferred into separate brown vials.
2.5.5. Characterization of pure compounds by nuclear magnetic resonance (NMR)
The clean samples were sent to Mr F. H. Makhubela of the Chemistry department,
University of Limpopo (Medunsa Campus). 1 H NMR and 13 C NMR spectra of the two
purified components were run with Varian Gemini 300 MHZ NMR Spectrometer (Merck)
at 300 MHZ using acetone as the solvent signal reference. The spectra analyses and
structure elucidation was performed by Dr L. Mdee of the Pharmacy department,
University of Limpopo (Turfloop Campus).
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