I have a good separation of nucleotides on a c18 with tetrabutyammonium ions as ion pairing agent but have a problem that the column so easily get overloaded and the largest peaks get very broad at the same time as the smallest are just above the detection limit. Does any one know the reason for the low capacity? For comparison, I have never had this problem on ion exchange columns although I load ten times more. I also wonder if there is any solution for this problem on reverse phase columns. I have tried mixed mode columns (RP with ionic groups) but then the nucleoside triphosphayes became extremely broad instead. Does the choice of ion pairing agent affect the loading capacity for example?
Hi Anders,
have a look here: https://www.researchgate.net/post/Nucleotides_via_HILIC_Column
I've posted here the method I'm using for oligonucleotid separation. I'm using Hexylamine and Hexafluoroisopropanol as additives. Method works very good (see attached chromatograms). I do analytical and preparative separations. The preparative separations are coupled with an analytical fraction collector. As preparative column I use an XBridge column (dimension: 50 x 10 mm, 5 µm, 130 A).
Regards
Markus
Hi Anders,
related to your question I've found the following article:
http://quimica.udea.edu.co/~carlopez/cromatohplc/hplc_basics_ph_overlap_bueno-2006.pdf
Loading capacity seems to be a function of the mobile phase pH. Higher pH values results in a higher loading capacity.
Regards
Markus
Hi Markus, it was interesting to read the paper to get more understanding of what is happening. In my case, it will be difficult to lower the pH enough since the nucleotides are always negatively charged (the pKa is less than 1).
When it comes to your method I am curious what difference it makes on selectivity, peak shape and loading capacity if HFIP is there or not. I am also wondering about the same thing regarding using hexylamine vs tetrabutylamine. Have you ever tried nucleotides instead of oligonucleotides?
Hi Anders,
I've only analyzed oligonucleotides in the mass range up to 5000-6000 Da. I guess that nucleotides are more difficult to separate with an RP column. HILIC would be the better choice for this case. Maybe you can try it with an AQ column (100% water) or a very shallow gradient.
Regards
Markus
It works perfectly to separate the nucleotides, my only concern is that the loading capacity is so low. I am still a bit curious of the effect of HFIP though, does it affect your separation? Have you tried without also?
Hi Anders,
HFIP is used to enhance the ion signal intensity. Look here:
Article Systematic optimization of ion-pairing agents and hexafluoro...
It also improves the retention and the separation of the oligonucleotides because it affects the solubility of the alkylamine ion pair reagent:
https://phenomenex.blog/2017/03/01/how-hfip-makes-your-lc-uv-oligo-method-more-awesome/
Article Comparison of mobile-phase systems commonly applied in liqui...
Regards
Markus
Thanks a lot. It seems like it is mainly needed for MS detection then rather than helping the chromatography.
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