Chromatography Forum

Hello,

I've running some ion-pair experiments on synthetic oligonucleotides (~25 bp) using this Technical Note from waters. I'm sure at least one of you knows it.

https://www.waters.com/webassets/cms/li ... a20770.pdf

The HFIP/TEA gradient works very well (separated from N-1 to baseline) but HFIP is very pricey.

I tried the TEAA gradient but I can't achieve the resolution. I was wondering if anyone else had done any similar work with these ion-pairing systems and could recommend anything?

Columns utilizing non-porous poly(styrene-co-divinylbenzene) stationary phases like the DNASep column from Transgenomic achieve n, n-1 base separations with TEAA. Other PS/DVB columns like Hamilton's PRP-1 are also great for oligo separations. Efficiency on this column is lower than DNASep due to the larger particle size (PRP-1: 5µm, DNASep 2.1µm).
Be advised that with both mobile phases (TEAA and TEA-HFIP) the separation is not only dependent on length but also on base composition (T being more hydrophobic than G, terminal bases have greater influence on elution order than bases located in the middle of the strand.
Separation performance usually increases at elevated separation temperatures (60-90°C are recommended).

Use of tetrabutylammonium bromide as ion-pairing reagent suppresses base dependent separations but it is not volatile (problem with MS).

Check out Transgenomic's web site for more info. Not sure if they sell the DNASep without the instrument but it is worth a try.