Chromatography is an analytical technique commonly Used for separating a combination of chemical compounds into its individual parts, so the individual parts can be completely examined. There are various kinds of chromatography e.g., liquid chromatography, gas chromatography, ion-exchange chromatography, affinity chromatography, but all of them employ the same basic principles. Chromatography is a separation technique that each Organic chemist and biochemist is acquainted with. I, myself, being an organic chemist, have routinely completed chromatographic separations of an assortment of mix of compounds in the laboratory. In actuality, I was leafing through my study slides and came across a pictorial representation of a real chromatographic separation I had carried out in the laboratory. I guess that picture would be a great starting point for this tutorial!
Let me first clarify what I was trying to do here. I had two reactants ‘A’ and ‘B’. I let them respond with one another, under specific reaction conditions, to form a products. After the reaction was complete, I wound up with a reaction mixture that contained unreacted A, unreacted B and my preferred product C. My job was to separate out A, B and C to isolate and examine pure merchandise C. First, as shown at the left hand panel, I conducted a thin layer what is chromatography TLC plate. This is essentially a rectangular piece of glass plate, coated with a thin layer of silica. I implemented a place of the reaction mix just above the bottom of the plate denoted with a solid line, and put the plate in a jar which contained a suitable organic solvent in this case, 1:1 volume by volume mixture of hexane: ethyl acetate was used, with only enough volume to dip the lower border of the plate. Gradually by capillary action, the solvent began rising up the silica plate, and as you can see the reaction mixture split into 3 spots with different color by the time the solvent had attained the solvent front markers.
Next, in order to actually perform the separation, I constructed a glass column as shown on the right hand side of this picture. I Took a glass column with a stopcock attached at the base, inserted a cotton Plug at the base of the column and packaged the column with a slurry of silica Gel prepared in an organic solvent. When the column has been packed, and the Solvent volume over the bed reduced to less than 5 mm, I carefully poured the Reaction mix above the bed of silica from the surface of the column, with the aid of a glass pipette. I opened the stopcock and Allow the solvent run slowly through the column. I always kept adding solvent from the top of the glass column. As you can see, the reaction mix began dividing into three Different bands – yellow, orange and pink equivalent to unreacted B, unreacted A and the desired product C, respectively. I accumulated individual bands in Separate flasks and was thus able to get pure C!