[biocat88]

I loaded my samples with mixture of glucose and galactose at the first time (developing solvent: ACN/H2O=85:15; color reagent: DAP; left picture in attachment). It looks that the separation is quite good, and my samples only contain glucose. But when I did the second time (right picture in attachment), I found that my samples have tailing problem, and my samples contain both galactose and glucose. I am really confused.
I am wondering is it possible the composition of developing solvent was changed because of less tight developing chamber, and it will cause the tailing problem?
Many thanks in advance!

Biocat

[Doc Oc]

Your samples might be too concentrated.  I'm guessing you're spotting fractions from a flash column.  How are you spotting them?

[biocat88]

Your samples might be too concentrated.  I'm guessing you're spotting fractions from a flash column.  How are you spotting them?

I loaded the same amount (0.5 ul) of sample by pippeting like the first picture.

[fledarmus]

What solutions are your samples in? If your knowns are in acetonitrile, for instance, while your flash column fractions are in acetonitrile water, you may end up with your fraction spots essentially in a water solution as the acetonitrile evaporates. This can lead to tailing of the fraction spots that you wouldn't see in the knowns.

[biocat88]

What solutions are your samples in? If your knowns are in acetonitrile, for instance, while your flash column fractions are in acetonitrile water, you may end up with your fraction spots essentially in a water solution as the acetonitrile evaporates. This can lead to tailing of the fraction spots that you wouldn't see in the knowns.

The solution of my samples are water. After spotting my samples on TLC plate, air dry step was always followed to make sure no water left before developing.

[biocat88]

I diluted one of my sample, and find that diluted sample looks better (see in the picture). I guess probably high phosphate concentration (500mM) in my sample make the tailing problem. How do you think?