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- Posts: 1
- Joined: Fri Sep 16, 2022 2:11 pm
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