I tried to work using same method I have worked before. But now not getting any peak. I am confused. Unable to understand what's happening. What can I do now?
You need to do some troubleshooting. There is not enough information to do more than suggest a very broad troubleshooting procedure.
What do you mean "not getting any peak"? HPLC problems fall into general classes:
- Pump- are the pumps primed? Are they running correctly?
- Injection - Is the injector working correctly?
- Detection - Is the detector working correctly?
- User - Are the correct solvents chosen? Are the solvent lines in the correct bottle? Is the correct column being used? Was the autosampler programmed correctly? Correct method chosen? Are the solvents mixed correctly (if needed for the method)?
There was nothing wrong with pumps, detector, injector. When I have used c8 column for another product pumps, detectors and injectors was functioning properly. But when I used c18 column again for some product dramatically there was no peak.I have used three c18 columns but the result was same.
First day retention time was shifting but from third day showing nothing.
First, run a reference. This should be done any time a change is made to a chromatography system. A reference is simply a compound that runs in the solvent system used for the column that verifies all major components (including the column) are working, and also the change was made correctly.
If you know the system is working, you can then look at your compound and method. Is the compound stable? If the retention time is shifting, either the chromatography method isn't stable, or the compound is changing.
Are you saying that before you were getting peak for you analyte(s) of interest with C18 and now you are not? And if you tried 3 different C18 and did not get peak(s) but with C8 you got the peak(s)? This does not quite add up.
Not for the same analyte. I have used c8 for another analyte. So I think if c8 column is working properly pump, detector and injectors are working properly.
When you ran the C8, everything was working fine at that time and for that column.
Does your C18 method use the same solvents and solvent modifiers as the C8 experiment? If not, were the solvents changed correctly? Lines in the correct bottles? Pumps primed? Just because the system worked during the last experiment doesn't mean it is working now, especially if something has been changed. That's like boarding a plane, and the pilot tells you s/he doesn't need to do a pre-flight because the plane was just working fine during the last flight! In high-throughput analytical labs, they will periodically run positive and negative sample to verify everything is running correctly.
Is it possible your analyte isn't in your sample? The only way to be sure is verify with some standards that the system is properly running.
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