Problem with Column Chromatography

[Jun]

Hi there. I am recently facing some problems with my experiment.

I am trying to fraction a natural plant using the column chromatography. I had been carried out this fractionation for several times by using the same conditions (same column,solvent, silica powder,but different batches of my sample). Recenlty I am trying to fraction this plant again but one of the fraction is missing during the column chromatography.
I had run the TLC for this crude sample and i found that this crude sample has the same profile with the previous batches of sample. The only different is that the color of the missing fraction is slightly lighter that the previous batches of sample. Will this influence my result?

Besides that just want to know whether a new column will influence the result too?

Thanks for the patient to read my problems. I hope some one can give me some advice and idea.