please explain more about the eluent (mobile phase) which this fraction is obtained by that. Also, how many spot was observed in TLC for this fraction?
The purity of a material eluting from a chromatography column is dependent on three parameters - the k' which is how long the material is retained on the column, "alpha" which is the difference between the retention of all of the other material and N the number of theoretical plates in the column. You sometimes need to adjust all of these parameters to separate complex samples but there is a methodical way to go about this so that you end up with the desired separation. There are many books on the subject.