Chromatography Forum

Hello! I have a problem. I am performing simultaneous quantification of vitamins A, E and D and last week I was able to separate all the peaks. The working conditions were:
mobil phase hexane:isopropanol (99:1), 1ml/min, 290nm. The column is Nucleodur NH2.
This week I tried again and now ganma tocopherol overlap with Vitamin D. I have repited sevaral times and it's the same. I have equilibrated the column for 2 hours before the firs running and I have made fresh solutions. Does anyone knows what is the problem?

Thank you in advanced

hi, how you prepare vitamin E stock solution from viscous standard.

Is the overlap due to wider peaks or to a change in retention?

If it was a change in retention, did all your peaks shift or just one of these?

Wow, I haven't used "normal" phase in three decades. But I remember that the trace water picked up by solvents could be important to causing irreproducibility.