I am developing a method for the analysis of folic acid in a supplement tablet. It is based mainly off USP method for tablets. Peak shape and resolution from other components are great.
The problem is that retention time varies greatly will do so in an unpredictable manner. The peak area is not affected by this shifting. This only happens when injecting samples pretty steady with standard at same concentration.
Any ideas?
thanks

For the most part, retention time reproducibility problems are pump related. Drifting retention time can be a sign of chromatography problems but "scatter" in the retention data is a sign of a problem with either flow rate or eluent composition. Is there a pattern to the "unpredictable" behavior? For example, are most retention times in a normal range with occasional "dropouts" exhibiting longer retention time that varies randomly? This would be indicative of a check valve problem. Other possible causes of reproducibility problems are: eluent outgassing in the check valve, particulate laden eluents, inadequate pressure on eluent bottles (manufacturers often claim that their instruments work fine without any pressure on the eluent bottle but invariably the best reliability is achieved with a pressurized eluent source), a failed on-line degassing unit or a malfunctioning proportioning valve.

Chris - flow rate does not appear to be the problem since he says that the peak area is consistent

Thanks for the replies,

There is not a pattern in the retentions times. it may start at nine minutes go up to 12 mins and back down again. Sometimes you will get the same time for consecutive injections next injection may differ by up to a minute. But peak shape is always very nice.

I believe particulate may be a contributor, I filter with 45 micron it is slow (filtrate appears clear) I tried 20 micron but filter clogs quickly, I may double filter.

Chris can you explain to me why area is expected to change with change with flow rate?

Thanks Lee

I guess peak area depends on analyte concentration + time spent in detection window.
Am I right ?
Jan

Lee,

Peak area is based on time vs. response with conventional data system output. If there is a major decrease in flow rate, the peak will take longer to pass through the detector and hence give more area counts. If the area were based on the volume vs. response, there shouldn't be any dependence on flow rate. In my discussion above I hadn't ruled out the possibility of checkvalve problems causing flow variability in spite of the area being consistent because I wasn't sure of the magnitude of the retention time variability and there was no way of knowing whether or not the flow rate variability is progressive or "digital". Theoretically, flow rate can fluctuate without area changing as long as the flow rate is constant during the time when the peak passes through the detector. For example, if there were some obstruction in the injection valve which was intermittent and the pump has some sort of "pressure feedback" control loop, then the flow rate might be correct when the peak is passing through the detector but lower for variable amounts of time immediately after the injection. I have seen extreme cases of this here in column manufacturing while testing short columns. I can't say for sure whether the area was consistent in this case but theoretically, anyway, it could be.

Ok I see

I’m not a big hplc guy but I was thinking like this: lower flow rate gives broader peaks with less peak intensity. absorbance being proportional to concentration this means the detector sees less analyte at a given time but overall the same amount of material passes though the detector for analysis. Molar absorptivity is constant and if we are working in linear range of the detection system area integrated area would be equivalent.

So it is the low rate of sampling utilized in HPLC detection that causes the area difference?
Lee

This has been discussed in detail before. Anyway: Think about what happens if you stop the flow when part of the peak is in thre detector. If you let this stop-time approach infinity, your peak area, obviously, has to approach infinity. A bit drastically put, but...