There are lots of constituents in the plant extract. It is difficult to apply a single separation technique to isolate individual compounds from this crude mixture. Hence, the crude extract is initially separated into various discrete fractions containing compounds of similar polarities or molecular sizes. These fractions may be obvious, physically discrete divisions, such as the two phases of a liquid-liquid extraction or they may be the contiguous eluate from a chromatography column, e.g., vacuum liquid chromatography (VLC), column chromatography (CC), size-exclusion chromatography (SEC), solid-phase extraction (SPE), etc. The general features of the molecule for the isolation process include
Solubility (hydrophobicity or hydrophobicity),
Acid-base properties,
Charge, stability, and
Molecular size.
If you going to isolate unknown compounds, it is advisable to carry out qualitative tests for the presence of various types of compounds, e.g., phenolic, steroids, alkaloids, flavonoids, etc., as well as analytical thin-layer chromatography (TLC), or HPLC profiling. The nature of the extract can also be helpful in choosing the right isolation protocol. For example, a MeOH extract or water fractions from this extract containing polar compounds are better dealt with using reversed-phase HPLC (RP-HPLC). Various physical properties of the extracts can also be determined with a small portion of the crude extract in a series of small batch-wise experiments. Some of these experiments are summarized below.
At the level of your inquiry, it is safe to say that don't jump (yet) to sophisticated techniques like HPLC, HPTLC, LC, MS, GC, and whatnots. Run a column chromatography.
Get ready with simple glass columns, silica gel of different mesh sizes, and solvents. And TLC should be made ready for testing the fractions. It is only from the most relevant fractions that you can separate and isolate the compounds.
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