Strange Column Overloading Behaviour

[M Farooq]

Is it possible that during column overload, the analyte peak elute at two different points? One peak is at the dead time, and the other at its specific retention time, say a k = 10, as seen under analytical conditions? One usually expects nested tails as one overloads the column. In another case, one may have a flat top due to the saturation of the detector, of the retained peak. However, in this case, the dead time peak has a flat top, and the retained analyte peak has also increased in size, surprisingly, not proportionally.

The NMR analysis shows that the peak eluting at the dead time and the retained peak are the same thing. Is this possible? One would expect the analyte peak would elute earlier and earlier as we increase the mass overload. The column is a reverse phase material with a ACN-H2O-TFA system as an eluent, and the analyte is a hydrophobic ligand with a positively charged metal center.

Thanks.

[Gerhard Kratz]

I guess your pH is around pH2. Part of your analyt may have changed from positively charged to neutral. What is the pK?

[danko]

It reminds me of something called ”break-through” but not exactly.
In such a case the load is so huge that all the stationary phase (throughout the whole column) is “covered” with the analyte causing some of the sample to just travel through the column completely unretained.
Maybe – instead of too much thinking – you should just load 10 times less sample and see what happens. Then do return and tell us what you saw.

Best Regards

[M Farooq]

Thanks. The breakthrough was my first thought as well, but breakthrough curves look different. However in this case, there is a baseline, and the peak of the same compound several minutes apart. I have never seen this behaviour before. Under lower mass load the chromatogram looks like a normal one. Note that in some rare cases, overload peaks split into two identical peaks shapes, but those peaks are not completely baseline resolved. That was seen on a column which had two different functional groups.

[Peter Apps]

Are you increasing the injection volume, or the concentration ? And is you injection solvent stronger (more organic) or at a different pH than the mobile phase. With a big enough injection volume and poor mixing you can get a slug of unretained analyte carrying through the column in plug of mobile phase enriched with injection solvent. This usually gives only a shoulder or a partial peak split.

Peter

[M Farooq]

I guess it is the injection solvent effect. The injection solvent is methanol while the mobile phase is ACN -water-TFA.