<italic>I did receive a PTH column but sent it back just before they
went onto backorder because the resolution between Q/T and K/L didn't
look right. I did manage to get another column sent and it too gives a
similar profile. The Q/T separation is not great.
</italic>>
It all depends on what you call resolution. If your peak widths at half
height for Gln and Lys are normal, then it's probably a positioning
problem. You can modify the temperature by 1 degree at a time to move
Lys around (increasing temp. brings PTH-Lys off earlier and vice
versa), or you can increase the steepness of the gradient slightly at
the end step of the chromatography. As I recall, PTH-Gln's position is
senstive to the concentration of THF. I can't remember which way it
moves, but if you have to increase it, you might buy a bottle of 5% THF
from ABI if they still sell it and mix it with buffer A. I'd make small
changes around 1% and test each time.
<italic>Is it true that if a column is on its way out then the
resolution between Q/T and K/L are the first ones to go?
</italic> What we usually see when a column is going is increased
interaction between the basic PTH's and exposed silanols of the column.
This requires either increasing the ion strength or the pH.
Alternatively, you can drop the ion strength and pH to reposition His
and Arg to the right of Ala and Tyr respectively.
Hope this helps.
regards,
Gary Hathaway