**5. Further fractionation in column chromatography**

The most potent fraction is selected and subjected to fractionation in column chromatography using silica gel. The column fractions are eluted using hexane, hexane ethyl acetate, ethyl acetate, ethyl acetate methanol, and methanol (in order of polarity) as mobile phase. The obtained pure compounds are isolated and identified through thin layer chromatography (TLC) and functional group analysis by nuclear magnetic resonance (NMR) spectroscopy [26, 27].

A glass chromatography column is set up. A piece of wool is placed in the bottom of the column and tamped down with a glass rod. The column is attached to a clamp stand and securely fastened in a vertical position. The column is then filled with hexane. Some (about 20) grams (20 g) of silica gel (60 mesh) is poured into a flask containing hexane; the slurry (mixture of hexane and silica gel) is then packed into the column while tapping the glass. After packing, the excess solvent is drained (using the tap at the bottom of the column) until it just reached the top level of

#### *Pharmacognostic Study of a Plant Seed Extract DOI: http://dx.doi.org/10.5772/intechopen.81860*

the silica gel. A thin layer of cotton (adsorbent) is placed on top of the column to prevent it from being disturbed when fresh solvent is added. The setup is ready for loading of the choice fraction with the best bioactivity. In the case of this research, the chloroform fraction was chosen. A quantity (say 2 g) of the chosen (chloroform) most bioactive fraction is mixed with little quantity (2 g) of silica gel and left to air dry.

 The chloroform fraction and silica gel is loaded dried to the top of the column. A small amount of the eluting solvent (hexane) with which the setup was loaded is added and allowed to drain in until the mixture was a little way into the adsorbent (cotton), and then the column was filled to the top with eluting solvent (hexane). The solvent system, starting with 100% hexane and 0% ethyl acetate, with subsequent increase in the polarity by 1%, is added. The eluent (fluid/solvent mixture) is collected in numbered test tubes of 15 ml each from the tap below. The column fractions are further eluted using hexane ethyl acetate (i.e., 99–1%…1–99%), ethyl acetate, (in order of polarity) as mobile phase. This was done by increasing the polarity by 1% alteration in eluent solvents' ratio (i.e., ethyl acetate 100%; ethyl acetate 99%/methanol 1%; till methanol 100%) [27]. The process is guided by thin layer chromatography (TLC) monitoring, for an effective separation in the eluent (solution containing "pure compound"). The procedure was stopped on eluent of the last pure compound. The solvent level is never allowed to drop below the top of the adsorbent. The eluent with the same bands of compounds are pooled. It should be said that the researcher could at this stage harvest pure compounds, seen in the TLC as single band. The process is discontinued when the compound(s) desired are off the column. The eluents collected in pools are put in the rotary evaporator and run to harvest the compound (pure or in two or three).
