Chromatography Forum

My PI and I noticed that our standard metabolic panel which is ran on a 36 minute HILIC Luna NH2 column with 5 mM Ammonium Acetate (pH -> ~9.9) & 100% ACN. We have a really hard time seeing any dNTPs at all. Could this be due to the column chemistry we are using or potentially our gradient elution method?

Does anyone have suggestions for optimization or do you believe it might require a different column and or buffer setup to achieve this?

There's a wealth of examples of AMP, ADP, and ATP as well as DNA or RNA oligonucleotides, being negatively impacted by metal surfaces in the HPLC column (and system). Options are PEEK columns or coated columns.

Here's a nice study concentrating on HILIC separations of oligos and the impact of low adsorption surfaces: https://www.sciencedirect.com/science/a ... 7322005179

The pH was lower than what you're using, and these interactions are exacerbated at lower pH, but still present in some respect at the high pH.

After this was published (so the column wasn't available to be included in the study), YMC also released a coated HPLC diol column. That data can be found here:

https://ymc.eu/files/imported/applicati ... -HILIC.pdf

Might be something to consider.

Yes, phosphates are a pain and stick to steel surfaces. My feeling is that the Luna amine column also isn't all that great; it's quite an old hilic column now, and seems a bit delicate and unreliable compared to some of the more modern columns.

But if you're looking at dNTP's, it's also worth considering an alkaline-tolerant C18. If you can run the samples in mildly alkaline conditions, you can often get enough retention to be useful, and C18 is just so much more robust and predictable. These are also available in coated, non-phosphate-sticky versions.