Chromatography Forum

I had been using tris buffer and sodium phosphate buffers at ph 7.9 on my anion exchange column. I could see few clear peaks in blank runs ie with out any sample injection. I strongly suspect the reagents as we ruled out the chances of involvement of the column or LC system in this problem. Previously I was using reagents sodium phosphate and tris from other brand, I tried your 99.9% trizma. Now the peak area and peak numbers have come down, but still could see clear peaks. Can I ask you if you have come across such a problem asked to you before and if do you have a better grade tris and Nacl (in eluting buffer) with possible little impurities that are not picked up by anion exchangers.

Have you dismissed the possibility of contaminated water?

Best Regards

Thank you for replying. Do you think its because of nucleotide materials in water? I did try to check the spectrum of milliQ purified water I use. It did not show me any contamination at 250-260nm wave length. The problem is I use the water from same sort of purifier (milliQ - brand) to blank the uv spec. Is there an other way to check it.

regards,
jith

Is there an other way to check it.

Yes! Just use the water instead of eluent A. Then run the gradient program once with the normal equilibration time, then equilibrate for much longer (3 – 4 times the normal) and run the gradient again. You should not inject anything – just zero mL.

If it’s the water you’ll get much larger peaks when running the gradient following the longer equilibration time.

Best Regards

Hi there,

I did long equillibriation buffer A, the ghost peak signal increases with increase in equillibriation time. I will do the same with water, thank you very much again.

jith