I am working with HPLC with ECD detector for neurotransmitter estimation from rat brain homogenate.. Few days back when i was doing HPLC, the base line reached from 2.5mV to around 1000 mV suddenly, then detector showed out of range.. Then i was washing column with methanol for next three days, still that problem persist. what would be the reason for that and how to solve this problem.
Thanks in advance for your valuable time and suggestions.
Washing a column with MeoH for three days really makes no sense. Why were you doing this? If the column is fouled, then wash it with as a strong solvent, that is appropriate for the column and samples used to elute off ALL retained material. Your method may be insufficient to elute the material being injected or your samples may be clogging the system.
Things to consider: Start with the most basic things. Probably not the detector, but easy enough to check first, (1) Are their any leaks; (2) Are their any air bubbles in the cell or flow path (air = bad); (3) Check the potentials and background currents on the cell; (4) Did you clean the column off (completely) and then re-equiibrate it in mobile phase? If so, did you inject a blank and look for the Tzero mark to confirm operation? (5) Are you using fresh mobile phase?; (6) Is your column fouled? (7) Is your backpressure steady?
Try and provide more information about your method, instrument, detector type/brand, all conditions and your steps to troubleshoot for more help.
Hello Letter!!!
Thanks for your valuable suggestion Bill Letter...
For brain homogenization we are using 17% percholic acid. For 500 ml mobile phase contains, citiric acid 3.76g, diasodium hydrogen orthophosphate 0.8872g, octane sulphonic acid 0.054g, EDTA 0.0092 g, methanol 90 mi and double distilled 410 mi..
We are using freshly prepared mobile phase for everyday.. We followed same procedure before it has come properly but now only it happening this kind of problem.
We are using Antec detector, sillica C18 column..
We are using methanol for column elusion what would be your suggestion for strong solvent..
(1) Replace the column with a new one. Columns are consumable items.
(2) Try ACN / H20 (97:3) instead of MeOH as a wash solution. A bit of acid may help too (~pH 3). *You want to use a wash solution that has been shown to FULLY dissolve ALL samples into solution.
Hello Bill
We tried with new column also but results the same..
Sorry to hear that swapping the column with a new one did not cure the observed problem, but that is part of the troubleshooting process. Please refer back to my earlier suggestions. Did you confirm that the ECD unit is operating properly? When in doubt, stop running your sample and setup a test method instead. Confirm correct operation of the detector and HPLC system. You need to start ruling out what is working from what is not.
Hello Bill
That problem has been solved somewhat, that was due to some setting has changed, but still the problem has been persist.. Now base line is coming properly but now problem is that no peak is coming.. would you please provide me the values for the following.. what is the value for Range, Vout and Ec.. our settings I have attached here as image
Hello Bill
That problem has been solved somewhat, that was due to some setting has changed, but still the problem has been persist.. Now base line is coming properly but now problem is that no peak is coming.. would you please provide me the values for the following.. what is the value for Range, Vout and Ec.. our settings I have attached here as image
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