Chromatography Forum

Hi!

I am using a SEC column (mAb PAC SEc-1 from ThermoFisher) to analyse an IgG sample but I am always having problems with the first injection from each vial: The peaks have lower intensity than the ones for the following injections.

Has anybody had the same problem?

My system is an Agilent 1260.

Thanks in advance.

I've always recommended that the first injection of the day is a "throw-away" to clean up / condition the column. In this case I suspect that the column has some level of active sites which are strongly (but not irreversibly) binding and/or denaturing your protein(s). Those sites get passivated by protein from the first injection. That bound protein gradually comes off, so the process repeats each day. If the problem has started only recently, it may be a hint that your column is nearing the end of its life (some of the bonded phase has been lost).

I've always recommended that the first injection of the day is a "throw-away" to clean up / condition the column.

I used liquid and gas chromatography for 40.5 years, and we ALWAYS ignored the first injection of the day.

Once computerized systems came about, on our Agilent systems we made the first line of the Sequence Table as "Replace" (replace the value in the computer memory) with 2 injections, and then the second injection would replace the first. Second line in Sequence Table was "Average" which would average all that line's injections with the final injection on Line 1.

Am I understanding correctly? You are doing multiple shots from each vial and the first is lower than the rest.
What is your injection volume and what type of caps do you use?

The problem with the first injection from each vial is that the septum encloses too tight to the injector needle, which causes underpressure in the vial when sucking the sample fluid. This results in excessive resistance to the suction which in turn results in less withdrawn volume. The following injections do not suffer from that because the gap in the septum becomes larger for the following injections and the negative pressure is offset.
The solution is to pre- pierce the plug / septum before placing the vial in the autosampler.

Have a great time.