HILIC: Issues with adenosine, adenine and AMP carryover

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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Dear All,

I am running HILIC and I have a problem with carryover of adenosine, adenine and AMP (Adenosine monophosphate). I looked for an answer in many places and tried some of the other suggestions, but I am still struggling. I would really appreciate any help and suggestions.

I am running a HILIC-based LC-MS assay for ATP and its derivatives. The column that I am using is:

Phenomenex Luna® 5 µm NH2 100 Å, LC Column 250 x 2 mm
A: 20mM Ammonium acetate + 20mM Ammonium hydroxide in 95% H20 / 5% Acetonitrile (pH 9.5)
B: 100% Acetonitrile
Flow Rate: 0.25 mL/min
Gradient:
t=0 A=15% B=85%
t=13 A=70% B=30%
t=20 A=100% B=0%
t=38 A=100% B=0%
t=40 A=15% B=85%
t=50 A=15% B=85%

The separation is looking good. However, there is consistent carryover for 3 molecules (adenosine, adenine and AMP). The carryover is about 5% to 10% of the size of the peaks in a test sample. Blanks and washes don't seem to reduce the carryover. We are quite certain that it's the issue with the column, not with the autosampler path.

I am worried that my column (aminopropyl-based) has irreversibly adsorbed these compounds. What I also don't understand is why adenosine and adenine (less polar), as well as AMP (polar) are affected, but other compounds are easily washed off.

I look forward to hearing from you and thank you for taking the time to read about my issue.

Kind regards,
Paulina
Hi Paulina,

I just had my Phenomenex rep in and asked him a little bit about your issues. What he said surprised me, but I also did a little research and found some interesting results... here's a paper I found: https://analyticalsciencejournals.onlin ... .200400027

Anyway, he described this phenomenon as potentially being from the stainless steel of the column interacting with your analytes. He suggested passivating the system to avoid these issues with phosphoric acid. The paper suggests doing the same, as well as switching your buffers or mobile phase additives to phosphate alternatives.
From what I recall him telling me was that the peaks will generally grow with the carryover until passivation occurs. Another alternative, which he said phenomenex actively does, is sell titatnium alloy columns rather than stainless and these issues are resolved. If you want I could give you his email! Or reach our to your phenomenex distributor and I'm sure he'll give your more clarifying information compared to someone who has just heard about the phenomenon like me.

I really hope this helps!
Tyler
I am not familiar with practical realization of HILIC on aminopropyl columns, but I recall that on bare silica one should not decrease organic component below ~50%. That's what most application notes suggest.

Second, can you try your separation at low pH, using ammonium acetate / acetic acid rather than ammonia? Retention will be less apparently, but the behavior of your analytes would change dramatically due to their ionization state which may affect their ability to linger in the column.

Lastly, adenine and adenosine are not metal sensitive, but AMP certainly is (as all phosphates). So the issue is unlikely to be caused by chelation, if all 3 are affected.
You can try a deactivated surface colunm as Waters Premier or can be added citric or oxalic acid in the buffer.
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