Establish a benchmarking method (and representative data) BEFORE the system develops a fault. This can be either a method you regularly use or a method specifically designed as a system checkout, but it must be 100% reliable. Obtain a comprehensive set of data (back pressure, repeatability, sensitivity, and noise) from this method on the system when it is working properly.
Don’t allow problems to start. Be methodical and meticulous in the way you set up the HPLC instrument and where possible perform preventative maintenance routines..
Clearly define the problem:
Compare a good chromatogram with the problem one.
Don’t forget the obvious or assume anything. Check EVERYTHING.
- Retention times: Do they change for all peaks (including T0) or only some?
- Peak height: Are they smaller, large, show poor repeatabilit? Does this apply to all peaks or only some?
- Elution order: Does this remain identical or does peak order change?
- T0 peak: If it is the same (e.g. k’ changes) it is unlikely to be flowrate related, (as this affects all peaks including RI). If it is just retained peaks affected, could be temperature or gradient related?
- Are there any extra peaks?
- Has peak shape/width changed?
Check the easy things first.
Check one thing at a time.
Keep a written note of every single thing you do as you will either quickly forget or doubt yourself.
Use your benchmarking method to establish if problem is due to either the instrument or the method (e.g. the sample prep, method conditions, column or analyst). For example, if your benchmarking method gives acceptable results, the problem is not related to an instrument fault..
Only take something to bits as a last resort or if you are reasonably sure this is causing the fault.
Don't forget to use the CHROMacademy troubleshooters :-)
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