ENO-20/30, Smaller Peak (small peak, less signal, weak signal) with Standard for Nitrite and Nitrate

1. Inject 10 µl of 10 µM standard from the manual injector, mix nitrite and nitrate. When using the manual injector, please set the autosampler injector at the Load position.  Confirm if the nitrite peak is higher than 16  mV. It is very important to distinguish the cause of the problem from the autosampler. Always use newly prepared standard solution, prepared on the same day.  If the nitrite peak height is less than 16 mV, please go on. If you find that the trouble is with the autosampler, please see the solution for the autosampler in the attached file. If only the nitrate peak is small and it is less than 12 mV, please replace the reduction column to a new one.   

2. Expand scale of Y axis of chromatogram to +/-0.1 mV range. If there is oscillation, it indicates a problem of reactor pump. In that case, real flow rate of reactor should be around a half of 0.1 ml/min. You can check at drain after stopping Carrier pump and collect Reactor in a gradient cylinder.  If this problem happens frequently, please add tubing that is 3 meters in length of 0.125 mm ID PEEK (red) to the outlet of the reactor. It works to prevent air generation in the pump. The normal setting of the reactor pump does not have back pressure. The back pressure given by this tubing will help to dissolve the air into the carrier. 


3. The carrier pump should read a pressure more than 1.0 MPa at 0.33 ml/min. If it is much lower, an air bubble in the reactor is likely preventing the flow. Open the drain of the pump and aspirate the reactor slowly with a syringe and set the pump to 0.5 ml/min and run for 5 min, then close the valve. While the valve is open and the pump is running, softly tapping the inlet and outlet of the pump check valves with a small wrench may help to remove the air. 


4. Absorb some of the fluid in the drain with tissue paper and wait for 5-10 min and see if the tissue turned pink with absorbing NO2 from the air. If it becomes pink, it should be an issue of connection between the ENO-20 and the data acquisition, Powerchrom, or the sample is not loaded to the manual injector. Please confirm if there is a clog on the drain of the manual injector (port 5 and 6).   


5. If the tissue does not change color, the correct reactor is not coming to the drain.  Please absorb reactor from the reservoir and do the same thing.  Even it does not turn pink, the prepared reactor does not work.  In most cases, hydrochloric acid concentration is too low. If the tissue becomes pink, it indicates the flow of reactor is not correct.  If this is the case, see next step.

6. If the baseline is totally flat without any peaks, there is the possibility that your data acquisition is not connected to the data acquisition system correctly. To check this, please confirm that all wires are properly and securely connected.  


7. If the ENO-20/30 has never been washed with methanol, especially the reactor line, the system background is too high to genetate peaks.  Please wash the system with methanol. 

8. The precolumn may trap bacteria and they multiply.  There are bacteria that metabolize nitrite and nitrate.  Please try to remove the precolumn and inject standards (never samples). Then the quality of the precolumn can be found. 


9. The injection loop of the injector can have the same issues with bacteria generation.  To identify the quality, compare two injection manners,  injecting a high concentration standard quickly, and then injecting a lower concentration to keep in the sample loop for a few minutes. Former shows peak, later does not.  

Derek is the author of this solution article.

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