By Andrea on Friday, June 4, 2004 - 06:37 am:

Hello! I'm performing SCX chromatography of PNA (peptide nucleic acids...very basic...).
My eluents are
A: Ammonium acetate 20mM pH 4.5
B: Acetic acid 40%
I use this method because I need MS (so no NaCl...)
My PNA elutes AFTER 100%B when I'm going back to A.
WHY?
Any help would be appreciated...thanks

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By MG on Friday, June 4, 2004 - 07:48 am:

I recently saw some similar methods at ASMS; requested reprints but don't have them yet.

Anyway, what happens if you increase the hold time at 100% B? Will the PNA eventually elute?

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By tom jupille on Saturday, June 5, 2004 - 10:09 am:

Remember that the gradient doesn't get to the end of the column immediately. The "end" of the gradient is approximately = tG + tD + t0
- tG is the gradient time
- tD is the "dwell time" (the time required to move liquid from the point where the gradient is formed to where it enters the column)
- t0 is the column dead time.

For example, at 1 mL/min, if you have a 2 mL dwell volume (a bit on the high side for a new instrument, but not outrageous), a 150 x 4.6 mm column (i.e., an internal volume of 1.5 mL or so), and a 20 minute gradient, the "end" of the gradient at the column outlet actually occurs at about 23.5 minutes. Anything that elutes before that is fine. If your peak is coming off later than that, then an isocratic hold at the end of the gradient would be a good idea.

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By Andrea on Monday, June 7, 2004 - 06:18 am:

Thanks everybody.
I'm performing some new run holdig the 100%B a lot more.
Thanks tom for your suggestion...
I'll let you know...