Help on HPLC

Zhifeng Qin zq4 at columbia.edu
Thu Aug 17 17:37:19 EST 1995


I ran into some difficulty in purifying a RNA oligo with HPLC. Since I 
just started to learn HPLC, I am wondering if I am just making some 
obvious mistakes.

All in all, we synthesized a 21 mer RNA oligo on the ABI machine. The
oligo is kind of special in terms that it had a fluorescein at its 3' end.
We then went about to label the oligo with rhodamine. After the labeling,
we try to used reverse phase HPLC to purify it. We used a 15cm C18 column
from Waters. It kind of worked, but on the denaturing gel the main
fraction always shows two bands. And the other strange things is that
while the same fraction from HPLC migrates as doublet, different fractions
from HPLC migrate the same on the gel. We try flattening the gradient and 
switching to a longer (25cm) C18 column, but got the same result every time.

I wonder if there is other type of column that will work better for us, 
or many it is the gradient? Any advice will be extremely helpful.




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