We are having an issue with an increase in area for repeat injection of a standard. If we repeat an injection of 20 standard injections the area will increase for the first 15 injections, than it will start to level off.

Now for the information.
We have injected blanks through out the analysis and have found no carry over.
This is an isocratic method.
We have gone through a cleaning of the column and started the analysis. Stopped the pump and restarted the next day with a repeat of the growing trend.
We have examined the sample using a diode array detector and do not see any degradation or coeluting peaks. In addition the spectrum stay the same.
The wavelength is at a maximum, not a slope.

Any help would be greatly appreciated. Thanks

Overlay the peaks to see if there's any broadening of them - Also, how constant are the peak heights adn retention times?

There is no peak broading, as you would see if the peak came out latter. The only broading is the small increase along with the height of each progressive peak. The retention time is consistent.

Evaporation of the sample solvent? If the sample is made up in a mixed solvent, it could level off.

One possibility is a secondary retention mechanism capable of "irreversible" retention, presumably with a very low level of such sites. If this is the cause, you can verify it simply making an injection with your analyte at a significantly higher concentration for your first injection (say 20X) followed by a few injections at normal concentration. If the cause of the problem is a secondary retention mechanism, these sites should be saturated after your first injection of concentrated analyte and following injections at normal levels will fall into line with your steady-state areas thus far only seen after numerous injections at the standard level.

Another possible cause is if your analyte is reactive in some way with something which might accumulate on the surface of the stationary phase. For example, if your analyte is a chelating agent, the behavior you describe is not atypical, especially when using a stainless steel instrument. Or, if your analyte is readily oxidized, a similar phenomenon is often observed due to absorbed oxygen (reversed phase columns are fairly efficient at concentrating dissolved gases out of the mobile phase when the water content is high) reacting with your analyte until your analyte has depleted the majority of the absorbed oxygen and your analytical system has reached some sort of "equilibrium" with the analysis. In cases such as this, you have to be careful to perform the analysis under precisely controlled conditions. Otherwise, for example, making injections at irregular intervals will often result in erratic peak area.

Did you ever inject a blanc after you reached the plateau (after 15 inj.)? No carryover?

Chris you have given us some good ideas I will need to try. HW Mueller we did inject blanks after the plateau was reached and they were clean. As for the evaporation of the solvent, experiments were used injecting out of separate sealed vials and the same phenomena. Thanks for all the help. Sorry for the delay in response. I had been called onto another project.

Hi Chris,

I have often observed "disolved oxygen" or "air" peaks. What is the retention mechanism for disolved gasses on reverse phase columns? Thanks.

Tom,

Mr Dolan has writen an article some months ago, where he talked about oxygen peaks. He said that an oxygen peak can be treated just like another peak in reversed phase mode. The elution can be adjusted with the adjustment of the mobile phase.

could it be an autinjector issue? Do you inject from one vial or from different vials? If its from one vial, than there might be reduced pressure in it until the septum is perforated enough to allow equilibration.

Tom,

As mentioned above, one can think of gas molecules as simply another analyte (which is moderately hydrophobic). The retention can vary significantly depending upon their "hydrophobicity" on reversed phase columns. Since there isn't a good general detection mechanism, HPLC is normally blind to gaseous impurities but with electrochemical detectors its possible to detect the retention of both hydrogen and oxygen by reversed phase and confirm elution of oxygen and hydrogen.

Alex we have done multiple studies on the vial. Injecting out of the same one and out of different ones. Either way we get an increase in area count over time.
As gas impurities or any other impurity goes the blanks injected during the series show no sign of the peak.
It appears to be a column issue but we have not been able to verify it or correct it.

Do you have two columns, preferentially a brand-new one and your current column? Would be interesting to see if both show it.

Just to exclude the column: Did you try the whole thing with the column replaced by a capillary and low flow? There will be no retention. As you are injecting a standard, it should work. And it is faster then trying different columns.

Uwe we did try a new column which produced the sampe results. Excluding the column is a great idea. We will try that as soon as I get time. Working on entirely too many products. Thanks again for all your help.