To separate the phytoconstituents of the given leaf extract using column chromatography.
column, adsorbent [silica gel], petroleum ether, chlorophyll extract, etc.
Principle of column chromatography
Chromatography represents a group of methods for separating molecular mixtures that depend on the differential affinities of the solute between two immiscible phases.
In column adsorption chromatography a solid stationary phase and a liquid mobile phase is used and the principle of separation is adsorption. When the mixture of components is dissolved in a mobile phase is introduced in the column and the mobile phase is allowed to run through the column where individual components of the mixtures more with different speeds depending upon the relative affinity towards the mobile and stationary phase. The components with a lesser affinity towards the stationary phase move faster and hence elute net of the column first, the one with a greater affinity towards the stationary phase moves slowly down the column and are at top of the column hence elute later. Thus the components as the mixture get separated.
Preparation of column
Take a column and pack the bottom of the column with glass wool or cotton and then pack the column with adsorbent by wet packing method and packing is allowed to set by mounting column withstand the filter paper disc is kept on the top of the column to avoid distance of adsorbent layer during the introduction of sample or mobile phase.
Separation of components
Open the lower knob of the column so that sample component. Start moving the adsorbent of the column and get separated into different bands according to their affinity towards the stationary and mobile phase the components with a lesser affinity towards their stationary phase moves faster and are at the bottom of the column. the components with a greater affinity towards the stationary phase slowly and will be at the top of the column. In between intermediate affinity, components settle down. The petroleum ether was used as eluting solvent each component that is chlorophyll and xanthophylls were collected separately.
The bands of xanthophylls and chlorophyll are were observed as distinct colored bands and were eluted with petroleum ether as eluent.
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