Chromatography Forum

I have a question considering overloading of a column. I have pretty concentrated sample (2 g/L) and I am making very small injections (10 nL). Do I overload column? My peaks tail. Would it be better if I was injecting bigger volumes with a way less concentrated sample? Let's say 5 uL injection of 0.004 g/L (the mass injected the same as in the above case). I am working with HILIC column (25cm, 4.6mm, 80A).
Thanks,
Anna

Anna,

From the injection voumes and sample concentrations you described, I don't think you either mass overload or volume overlaod the column. But I prefer using 5-uL injection of 0.004 g/L sample since most autosamplers or injectors will behave well. Also use mobile phase as the diluent. In some cases, peak tailing results from secondary interaction between the analyte and stationary phase, which can be oversome by using appropriate HILIC stationary phases and mobile phases.

What type of HILIC column are you using, neutral, ionic or awitterionic surface? And what is the nature of your analyte, basic, acidic or neutral?

Hilic column - TSKgel Amide-80 HILIC Column, so the stationary phase is mostly amide groups. It is based on polymer. I assume that amino group (-NH2) is cationic. As mobile phase I use mixture of (80/20 v/v) MeOH and buffer (pH=5 but in such a big amount of MeOH (80%) pH is shifted toward bigger values ~6-7). To be honest we use also CO2 in mobile phase (enhanced fluidity liquid chromatography). It acts as nonpolar solvent. My sample = cytidine (2 pKas, pKa1=3.3, pKa2=13.5). I assume that in this mobile phase (pH~6-7) it is positively charged (+1). Stationary phase is positively charged as well.
Sample is prepared in MeOH/Buffer solution (exactly the same as in mobile phase, except that mobile phase contains CO2).
BTW, not retained benzene tails as well. Is it a just a system characteristic? Thank you very much,
Anna

I'm not sure if a sub-critical CO2 system would be easy to model, especially as a HILIC system, and I would not be surprised if the CO2 interacted with the water. I would guess that the whole CO2 system is the cause of the tailing, unless you previously had good peak shape.

You could probably try the column in a standard HPLC system using a suitable HILIC mobile phase to ensure that the column provides good peak shape for your compounds, and then move the analysis to your system.

You could also contact Tosoh Bioscience support to ascertain if they have any useful comments.

Bruce Hamilton.

Thank you for hints.
We used reversed phase column (C18) for that system (of course there was different percentage of MeOH versus buffer) with CO2 and no such a tailing was observed. I figured out that this is the characteristic of the column. Packing heterogeneity probably causes tails.
Thanks,
Anna

Lots of things could cause tailing, and I assume the column works OK in normal HILIC HPLC mode, which may exclude packing issues - unless subjected to out-of-specification environments.

I'm not sure that a standard RP C18 column would be a good model for HILIC in your system, but it certainly does exclude many other possible systemic causes of tailing.

Bruce Hamilton

It won't be such a bad idea to make sure the column is working properly. You can do so by running vendor suggested column performance test on a "known" good LC system. It is possible somehow the column was damaged. Tailing of benzene suggests system and/or column problems. If you are sure the system works ok, it is more likely the column is faulty.