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- Posts: 1
- Joined: Sat Nov 16, 2013 10:09 pm
Hi, I am pretty new to using LC-MS/MS. I have a question about some issues I ran into. I have been analyzing gefitinib cell uptake samples and used dasatinib as my internal standard. The mobile phase I have been using is ACN and water with 10mM ammonium acetate, running at at gradient from 10 to 90% Organic in 5 min, then holding at 90% for an additional minute with 3 min post-run. The flow rate is 0.4 ml/min. I ran the samples with a C18 column (2.1*50 mm, 1.8 µm particle size).
Yesterday afternoon I ran about 20 samples using this method without any problems. Then I set up a worklist to run overnight with the same method. When I returned in the morning, I found that only the first 4 samples were completed successfully. The error seems to be that the pump pressure went out of range. So I turned on the pump, the pressure for running the initial conditions 10:90 (ACN to water) at 0.4 ml/min wants to go above 600 bar. So I decreased the flow rate to 0.1 ml/min, the pressure was like 200 bar.
Why yesterday afternoon the method seems to be doing fine, but only 4 samples in the overnight worklist was able to complete due to back pressure? The column does not seem to be completely clogged up since the mobile phase was able to run through this morning. I did protein precipitation with my cell lysate samples, so the sample should be quite clean. Any ideas? Thank you so much!
Yesterday afternoon I ran about 20 samples using this method without any problems. Then I set up a worklist to run overnight with the same method. When I returned in the morning, I found that only the first 4 samples were completed successfully. The error seems to be that the pump pressure went out of range. So I turned on the pump, the pressure for running the initial conditions 10:90 (ACN to water) at 0.4 ml/min wants to go above 600 bar. So I decreased the flow rate to 0.1 ml/min, the pressure was like 200 bar.
Why yesterday afternoon the method seems to be doing fine, but only 4 samples in the overnight worklist was able to complete due to back pressure? The column does not seem to be completely clogged up since the mobile phase was able to run through this morning. I did protein precipitation with my cell lysate samples, so the sample should be quite clean. Any ideas? Thank you so much!
