Chromatography Forum

I have experienced a strange thing with the EPA 545 Anatoxin analysis. I have been running it well for about 8 months and the other day it suddenly lost about 80% of the sensitivity. I cleaned the instrument and did everything I could to figure it out. My trainee had made up the mobile phase A which is 100mM Acetic Acid in H2O. After two runs with low sensitivity I made up the mobile phase and it was right back up to the correct sensitivity. After talking to her the only difference was I used the 10ml repeating pipette with plastic tips and she used a 10ml glass serological pipette, same bottle of acid.

I always have to make about 10 injections in which the sensitivity falls then levels off, which I think is the acid reaching a stable concentration after an initial loss because of volatility, but even after about a 50% drop it runs stable at a level that is almost 10x what we were seeing when she made up the mobile phase.

This one has me stumped. I can't see anything that should be in the disposable glass pipettes that would cause that much effect on the mobile phase. Anyone else ever see something like this?

I have been working on that method, and a version for surface waters, for a year now and I have yet to see a problem with anatoxin-a reproducibility. I tried decreasing the concentration of acetic acid to test robustness and found it wasn't as critical as I expected.
However I continue to see issues with cylindrospermopsin in surface waters and in local tap water. I am using the labeled internal standards from Abraxis so the uracil IS is gone.

Steve Reimer wrote:
I have been working on that method, and a version for surface waters, for a year now and I have yet to see a problem with anatoxin-a reproducibility. I tried decreasing the concentration of acetic acid to test robustness and found it wasn't as critical as I expected.
However I continue to see issues with cylindrospermopsin in surface waters and in local tap water. I am using the labeled internal standards from Abraxis so the uracil IS is gone.

For me the Uracil IS is quite stable, but the L-Phenylalanine IS for the Anatoxin is what is causing the problems. I put on a new fresh bottle of mobile phase and watch as the first few injections have dropping area counts. Once it becomes stable it can run two 20 sample batches non-stop.

What baffles me is how one of us can make mobile phase and get an 80% loss in sensitivity when using the same reagents. It has to be something in the pipettes but I can't figure it out.

I went and looked again at the pipettes and the box has a label that states "nonpyrogenic sterile". They are the Kimble brand labeled "orange" in an orange box. If they are simply in a cardboard box and labeled sterile, would that mean they must have some type of sterilizing agent present on them?

I have found when evaluating gauze wipes for our chemical warfare agent analysis that the additives in those sterile items are huge compared to the levels we typically look for.

If you get dirty surface water the uracil can be completely suppressed by biogenic material.