Hello, I analyse neutral (e.g. benzalacetone), acidic (benzoic acid, saccharine) and anionic compounds (cumol sulphonate, saccharine propan sulphonate). My mobile phase is 10 mM phosphate + 1.5 mM tetrabutylammonium hydroxyde, pH adjusted to 2.70 with phosp. acid. I work with acetonitrile in gradient. The column is Nova-Pak C18, now about 2000 injections, guard column replaced after 350 injections in average, in use about 30 months. The column was stored in 100% ACN while in not in use. Ret. times have not changed since installed.
Changes in response were very little during fist 24 months (checked by regulatory standards). After that response for acids decreased by 28% and for neutrals by 13%. So I recalibrated the column. Two months later it went back to the previous values and almost fitted the old calibration. This seemed odd so I checked autosampler for compression. It was OK. Again after two months I found the response for saccharine propane sulphonate decreasing. My question is: can it be caused by column aging? I'm a little bit confused because plate number remains the same, peak shape does not change. Chemicals for mobile phase from identical bottles for last 2 years. Shall I replace the column with a new one?
Thanks for help.

Are you sure the problem is not in your detector? Assuming you are using a UV detctor, if your detector wavelength is at a region of your analyte absorption curve where the absorbance changes rapidly with a change in wavelength, a slight change in the detector calibration could lead to a noticeable change in response.

Such a phenomenon is unlikely to be a column problem, especially when retention and efficiency remain unchanged.

Are this changes related with changing the UV lamp ??
Are the solutions always new or could be some degradation problems with the standard solutions ?

I think this not detector related problem. I recalibrated detector (Waters 2487) and the response is just the same as before it. The wavelenght is 254 nm, so I assume that it should be robust for compound with benzene ring (all of them). The detector has installed the original lamp (about 5000 hrs.) The energy decreases contantly but I do not see any significant noise on the baseline. All the standard solutions I have prepared fresh before every recalibrating.
I made 3 repetetive injections today and the results are (area counts): 146,0; 145,8; 146,3. So it does not seem to be injector related problem. Anyway, I will try to more diagnostics on it.
Thanks. V.

How do you quantitate - peak area or peak height?

I usually quantitate peak area. But in some cases it seems to be better to use peak height (inon chromatography with conductivity detector).

This could be an effect caused by small changes in the flow-rate. Did you repair the pump around the time that the change occurred?

Or small changes in the pH of the dissolution phase ? Any problems with pH-meter ?

If you find the response, please tell us.

Hello, I tried test flow rate dependence:
Flow (ml/min) Area counts
0,980 ***** 160,8
1,000 ***** 158,1
1,020 ***** 153,9
So I think the change is not flow rate dependent. I also tried water instead of buffered mobile phase and the response was just the same. But I found something interesting about mixing the stanrdard. When I mix benzalacetone together with cholorobenzaldehyde the response for benzalacetone is significantly lower and I see another peak. This phenomenon is also confirmed by GC (except that I do not see any extra peak).
OK, I have problem solved for one compound. I have one more compound (nonpolar) which has always stable response (LC and GC too). I suspect saccharin that the bottle was not shut properly and the water content could be different (I made imide form and I will try to work with it). Anyway there is benzoic acid which also has changed over time. Its response could depend on pH of mobile phase. I should test it. Thanks. V.

Again, the problem showed up today. Again I started checking components one by one. I installed a new column so I found out that response is the same. So I checked autosampler and found no problem. So I cleaned the flow cell with 6M nitric acid and the response raised by 15%. In last two weeks I analyzed very nonpolar compounds (such as nitrobezoyl derivates of nonionic surfactants) and the cell was contaminated with dirt. So I believe that the numbers I described previously were only silly coincidence. And, I think that my deuterium lamp is kind of old, now over 4900 hrs. Could anyone tell if I am right that low lamp energy can affect quantitation? What about a combination of low energy and dirt in the cell? Thanks. Vojtech

You would be better off to do a calibration just before your analysis. This would eliminate such problems.