Dear all,

we have been running a quite simple HPLC method consisting of a phospate mobile phase (50 mM, pH 2,15), a RP-column (TSK-gel, 4,6x250) and UV-detection at 210 nm.
We´ve been running for several months without problems but on a recent run the sensitivity dropped with a factor of about 3. Also, the retention time of our analyte increased about 0,5 min. The funny thing was that at the end of the run the sensitivity and retention time were back to normal. The following day a set of standards was run but then the sensitivity once again had decreased.
The column is washed with 25% MetOH between runs and regularily with 100% MetOH. The injection volume was checked and had not decreased.
Anyone have a clue to what could be the reason? Is it the injector or the column?

Thanks in advance / Mattias

Phosphate could be precipitating out at the beginning of the run if the column has a lot of MeOH. Try rinsing the column with your mobile phase minus the phosphate before introducing the phosphate to the system. This may have done a couple of times.

How long is the run time? Is the change significant? Did the peak shape change? If the peak was a small one and was made broader by eluting late it could cause a decrease in area count.

what is the injector?

Look at your column dead time t0 and calulate the K for your peak. Are the Ks the same before and after? Let me know and we'll figure the next step.

What exactly do you mean by "sensitivity", the slope of your calibration curve, the area response of your standard injections, or something else?

If your standard response has changed by a factor of three I think your problem is in your injector. I have seen worn injector valves produce symptoms similar to yours. If your system uses an injector valve try replacing the rotor and cleaning the stator.

Sometimes worn rotors can develop small channels that will allow leakage from one port to another, thus you could lose a significant portion of your sample and have a small decrease in retention time. Good luck.

I meant small increase in retention time not decrease.

Thanks everybody for your response,

I don't think phospate precipitation is the reason, we have rinsed the column extensively.

What I meant by sensitivity is that the peak heigth and area has decreased. It also looks like the chromatogram has been "smeared" to the right with broadened peaks and increasing retention times as a result. We also have noticed that just prior to injection the pressure of the system drops to zero and then it takes about one minute for it to regain the original pressure.
To me (by looking at Tom's comments) it seems most likely to be the injector, we'll have a technician come look at it.
But of course I'm no HPLC expert, any other ideas are welcome! :)

/Mattias