Isolation and characterization of allelopathic compounds by column chromatography and fraction collector LAMBDA OMNICOLL

Isolation, structural elucidation and chemical synthesis of allelopathic compounds from plants, fungi, and other microorganisms are a very labor-exhausting and time-consuming studies. However, the identification of highly potent biological structures (used as natural herbicides, medicaments, antioxidants, etc.) is worth the time and effort spent on it.

A routine study on the allelochemical structural analysis becomes difficult, when the structures are not identified in the crude extract, even after several preliminary steps to achieve the fractionation of crude extracts (by maceration, ultrasound or microwave assisted extractions followed by aqueous two-phases extractions, etc.).

The researcher will then remain with a large volume of solvent which may contain partial mixture of structures (with or without similar biological activities). Biological activity of the structures can be identified by the bioguided study and assays. This large volume of solvent should be split into smaller fractions using solvent mixtures with different polarities. Fraction collecting will help to isolate the most active allelopathic molecules for the desired / target biological activity.

Mostly, the silica gel liquid column chromatography is used for fractionation of the extracts. However, it is common to fractionate and collect eluates of about 65 - 200 fractions, in-order to achieve the most purified bioactive structure on each eluate. Manual collection of large number of fractions is the most time and effort consuming phase in the general research project, due to the high cost of the spectrophotometric fraction collectors on the market.

Further purification steps include thin layer chromatography, bioactive assays, preparative column chromatographies etc. and finally, a list of spectrometric assays for the highly active structures, in order to elucidate and achieve its chemical synthesis in the future.

The collection of 200 fractions or more can be easily automated in the most feasible way (if the proper fraction collector is chosen!).

Fractionation of the crude extract

Initially the crude extracts are separated into various discrete fractions containing compounds of similar polarities or molecular sizes by the column chromatography. In this chromatographic separation, about 65 fractions measuring 20 mL each were collected (time based) by the fraction collector OMNICOLL.

Thin-layer chromatography (TLC)

An aliquot of all the collected fractions were loaded on the activated silica gel TLC plates (20 x 20 cm). The fractions were pooled based on the spotting pattern and Rf values on the TLC plate. The pooled fractions were numbered (Fr.1 - Fr.8).

Preparative TLC or HPLC column chromatography

The fractions (Fr.1 - Fr.8) can be further purified by preparative TLC and / or HPLC column chromatography. Preparative HPLC will help in isolating a particular compound more accurately.

Characterization of the allelopathic compounds

The isolated pure fractions are then used for the determination of structure and also to check once again the biological activity.

Characterization of the pure allelopathic compounds are done by high-resolution IR, MS, ¹H and ¹³C NMR spectra.

IR spectra used to know the functional groups of the isolated pure fraction. LC-MS gives either empirical or exact (HRMS) molecular weight and NMR spectra reveals the C and H environments. LC-MS analysis helps to compare the MS pattern.