Correlation of Normal Phase TLC to HPLC Results in the Isolation of Chlorophyll Degradation Components Extracted from Algae and Extra Virgin Olive Oil
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.