Chromatography Forum

Hi All,
I am having a problem with the Dionex DNAPac PA100 regarding column lifetime. We are separating PEGylated oligonucleotides and in order to separate the N-1 and N+1 species we are operating at the temperature limit for these columns (90 °C). We have two products that we run at this elevated temperature and I was wondering if anyone had any tips or tricks to extend the column lifetime at these extreme conditions other than what is printed in the column care manual. What we see over time is a gradual loss of retention of the main peak (9.25 min RT vs. 7.75 min RT). The RT eventually settles, but the chromatography is very different with the loss of retention. Any help would be appreciated.

Thanks in advance,

RJ

RJ,
Thanks for your post.
The DNAPac column comes in two "chemistires" the PA100 and the PA200. The PA100 was introduced in 1991 and was the gold standard for oligonucleotide resolution for ~ a decade. In 2004 Dionex introduced the PA200 with slightly modified chemistry, specifically for improving column life.
An advantage of the DNAPac PA200 is that it employs a smaller bead size, so it tends to provide better column efficiencey, as well as stability. Note that there is a modest selectivity difference between the two chemistries, and this has been used to advantage in certain RNAi studies.
The DNAPac PA200 increases phase stability by ~ 9-fold (see Thayer et. al. 2005 Analytical Biochemistry 338: 39-47 for more information on these two columns).
I would recommend you use the DNAPac PA200 if you must work at temperatures above 60 °C.
The two DNAPac columns are highlighted in numerous nucleic acid applications that are appended to the Dionex Therapeutic nucleic Acids Application Notebook. That NoteBook is available at:
http://www.dionex.com/en-us/markets/biopharmaceuticals/nucleic-acid-therapeutics/lp-87167.html
Cheers,
Jim Thayer

Thanks for the updated information. I will definitely give the PA200 a try and see if the chromatography is at least tolerable. Thanks again for your help.