Chromatography Forum

Hi all,

We've been running a stability-indicating SEC-HPLC method on Agilent 1100 for the past 30 weeks or so; the samples are stored at 2-8C and appear to be fairly stable. I compared the total peak area (280nm) from different timepoints and found it to increase in a smooth function over time. For example, at week 0, the total area was 4000 mAU*s, whereas at week 30, it was 4400 mAU*s. Filling in timepoints in between (e.g., 3, 6, 12, and 20 weeks) shows that the increase in total peak area is somewhat asymptotic, so that a plateau appears to be nearly reached.

This might be a problem with recovery from the column, but I have tried a couple of different columns at the week 30 timepoint and found the total peak area to be ~4400 mAU*s. Therefore, I don't think it's a column issue.

I also don't think it's a sample issue because I also run samples stored at different temperatures (-70C, -20C as stable controls) and they all show the same smooth increase in total peak area.

Could this observation be due to lamp life? Can one unequivocally say that the older the lamp, the greater the peak area (assuming the sample remains unchanged)?

Thanks!!

That was just discussed, and: remember the ratio Io/I in Beer-lambert´s law.

OK, thanks HW. So, I'll check my lamp performance; however, it sounds unlikely to be the source of the increase in total peak area.

Do you or anyone else have other suggestions as to the source?

Thanks.

In what kind of vessel do you keep your samples?
could it be simply that the solution in which you kept your sample has been evaporating inside the vessel eventhou it was kept at low temp.?
on a 30 week period this assymptotic behaviour would look more logical to me. even the transfer to the gas phase in the free volume space can explain this.
also if i am not mistaking the Io/I in Beer-Lambers law come to compensate for the reduction in lamp energy. the signal is reported in relation to a consantly driftting "100% signal". what it does not atone for is the noise which constantly increases has the energy level of the lamp decreases. so in my view the lamp life should not be the reason for the elevation in the signal but a physical factor that would cause the sample to go throu some type of concentration process.