Isolation of high purity examples of naturally occurring compounds is a challenging
task for many laboratories accustomed to an analytical chromatography workflow. It
may be unavoidable, however, when one discovers that many obscure components are not
commercially available in sufficient purity for use as qualitative or quantitative
standards in mainstream analytical use. Further, when the components lending observable
biological activity in a crude extract have not been identified, the chromatographer
depends not only on achieving purification of potentially hundreds of compounds but
also on knowing that all of the components in the extract are being successfully recovered
from the purification column, and in the sample chemical form as were present in the
crude mixture. Thin Layer Chromatography (TLC) can be a useful tool for screening
crude extracts and predicting column elution. It can be more effective than High Performance
Liquid Chromatography (HPLC), and it is a relatively easy to use tool in the hands
of chemists who may not have formal training or experience with HPLC. Translation
of the selectivity, retention and resolution observed with TLC methods to an HPLC
preparative separation can be challenging. In the presented work, we used extracts
from algae and bulk extra virgin olive oil to isolate chlorophyll and degradation
components in the pheophytin, pheophorbide and pyropheophytin structural groups. We
elaborate the key elements of TLC that allow recovery to be assessed, and how a straightforward
conversion to usable HPLC conditions can be achieved. The combined use of UV/VIS Diode
Array detection (DAD), Evaporative Light Scattering detection (ELSD) and single quadrupole
MS allowed for an effective characterization of structure and purity for the isolated
target compounds.
