**6. Separation with preparative TLC plate**

 Finally the compounds in two or three bands are separated using a preparative TLC plate after collecting compound in the rotary evaporator. There are different types of preparative plates. The difference lies in the capacity of how much compound it can separate and whether it has a concentration zone or not. Though expensive, the relaying researcher advices for the preparative TLC plate with concentration zone and avoidance of locally made ones that may input impurities with poor separation quality. The quantity (concentration) of the compound to be loaded onto the preparative plate at the concentration zone is dependent of the type purchased. It should be stated here that the loading of the compound is the crucial skill for a successful separation. So if the type purchased can only separate 50 mg at a go, make sure you load say 48 mg for a successful uniform separation.

 In preparation one would have first found out the solvent system that would best separate the compound through thin layer chromatography (TLC) testing. This solvent system is what is poured into the preparative TLC chamber (made of thick glass) that can accommodate a 20 by 20 cm preparative TLC plate with concentration zone. The procedure starts with first weighing out 50 mg of the compound to be separated and making into a fine dissolved solution ready to be loaded onto the plate at the concentration zone. The loading is done with a pipette such that it is diligently spread uniformly onto the plate from one end of the concentration zone to the other, just avoiding the very edges (right and left). Allow the plate to dry after the loading. Prepare the developing chamber (thick glass preparative TLC chamber) by making sure it's clean and dry before pouring in your solvent system of choice for the separation. The solvent level in the developing chamber should not be higher than the level of the concentration zone (or the point of load of the compound). It

should be just below the loading point when the preparative plate is dropped gently into the developing chamber and allowed to separate for an hour tops. The time is guided by a virtualized capillary movement of the solvent seen on the preparative plate. The plate is removed from the chamber after completion of the capillary movement to almost the tip of the plate and dried at very low temperature say 40o C. The separated compounds are located in the plate by using an ultraviolent light source, and the positions are marked. Since the compound would have traveled the same distance on the plate, it is easy to mark the region and scrape out the position on the plate into a clean beaker. A solvent is then poured into the beaker to dissolve the separated pure compound. The content of the beaker is then filtered to collect the solution (solvent and dissolved separated pure compound) which is allowed to evaporate by low temperature heat of 40°C.

The described procedures are repeated until the researcher has the desired quantity (mg) for both bioassay and structural elucidation. It is advised that one should have secured a research grant because several repeat processes are expected before a reasonable quantity can be gotten that would warrant for a successful bioassay.
