Chromatography Forum

I am using a mobile phase made of ammonium acetate buffer and acetonitrile. I found the standard peak area count decreased with the age of my mobile phase. The peak area cound went back when I changed to a freshly prepare mobile phase. Anyone experienced this? how to explain it?

What is the pH of the buffer? How do you degas the mobile phase?

Some compounds have fairly strong changes in absorbtion coefficient as a function of pH and/or organic content. Phenolics are a good example. Usually this effect is accompanied by retention shifts when the average ionization of the molecule changes.

Thanks.

I also experinced this before when I worked in my prevous job. The buffer used is pH 9 and detected at 210 nm. The run can not be more 24 hr long because the bracketing std always failed the 3% recovery limit.

The current method using buffer at about 6. I checked the mobile phase pH. It didn't change. I degassed the mobile phase by filtering through 0.45um filter. No additional degas performed.

I also noticed, the peak area decreased much faster when I used a bigger container for mobile phase.

At pH 9, you could have picked up CO2 from the air, which then could change the pH and therefore the absorption. This would happen faster in a larger stirred vessel. It is still possible at pH 6, but somewhat less likely. On the other hand, ammonium acetate is not a buffer at pH 6...

Maybe you are losing ammonia from your buffer at pH 9. Why not measure the pH of the fresh mobile phase and then the mobile phase after 24hr of use under your normal conditions.

Measure the pH after the addition of the organic solvent. Measure it actually in your reservoir bottle when you only have a small amount left and a big headspace in the bottle. Since you are looking for a change, the interpretation of the absolute figures is of less importance.

Thanks a lot. these are the good explainations. I will perform your suggestions.

watch out your temperature change both MP and room

I had some problems whit it in the past, I've monitored pH and it's changing.
Chemically it's not a very stable buffer. I'd try to keep it as fresh as I can and monitor pH from batch to batch…