Chromatography Forum

Hello everyone,

I have been struggling with my SE-HPLC for over a week now and definitely need some help. I am trying to characterize PEGylated lysozyme with varying molecular weights of PEG on an Agilent AdvanceBio 300A, 2.7um, 300mm, ID 7.8mm column. The column is old but had eluted the compounds fine once with a mobile phase of 100mM NaCl and 25mM phosphate buffer. The next time I eluted these compounds using this buffer, the tailing of the peaks and retention time became much longer and so I did a cleaning of the column with an acidic buffer which improved it marginally however it was still severe enough that I decided to try other mobile phases.

I have trialled two other mobile phases now including 100mM sodium sulfate/100mM PB and 25mM PB/300mM NaCl. The story is the same for both of these mobile phases: The native lysozyme elutes fine but the conjugants do not elute at all and I get no peaks.

Has anyone done similar characterisation and can advise me on this? Is there more troubleshooting I can do or is it just a matter of buying a new column? I trialled two columns for this and the results were the same; although they were both old columns.

Thanks.

Hi WB,

I am sorry but i got a little bit confused on your post. let me understand better. First injection was ok... second injection had a higher retention and broader peaks... after doing some washing and trying other buffers the peak for PEGylated protein vanished never to return... Is this correct?

Can you describe more about your method, like temperature, flow rate, injection volume and analyte concentration and pH of the PBS you are using? Can you share what were the pressures you were seeing throughout the whole event (from first injection and beyond)? for the case where there is no elution, have you run a method that is long enough to see if it elutes next to the "high retention" you describe for injection 2 or at the "maximum retention of the SEC column according to the calibration curve from manufacturer? Can you please describe your washing procedures youu have used? Also, do you know what was it stored in (example: water, sodium azide solution,...)?

SEC columns will measure hydrodynamic radius, so a higher retention in comparison to what was originally seen could mean some kind of damage (physical or chemical degradation) to the macromolecule (in other words, macromolecule from injection two would have a higher retention than the one in injection 1). Is there any chance this might be happening? There are also cases of strong adsorption due to secondary interactions between analyte and matrix, but i have never seen a peak vanish, it usually gets very broad. for these cases a very small amount of organic solvent can be added (about 5~15 %), but pay close attention on the solubility of your salts!

i hope this helps.