Chromatography Forum

I've been working to develop a method that separates a borderline protein (8kDa) from its oxidative degradates. I had settled on a Jupiter Proteo column using the traditional 0.1% TFA in water and acetonitrile and had good resolution for about 10 days, a good cal curve and repeatibility. Then a colleague gave me a solution of the peptide containing sodium metabisulfite as a preservative. I did not see the peptide in that run. I did not see the peptide in a 24 hour hydrolysis sample which should still have nearly 100%. I went back and injected the standard I had run at the start of the day, but the peptide peak was not to be found. I've remade the mobile phase, changed the guard column, prepared a fresh standard solution, and rinsed the column in 80% water/TFA, 100% ACN/TFA, and in ACN:iPA 2:1. The peptide peak will not come back. There has been no increase or decrease in column pressure. A benzyl alcohol standard has good response and peak shape. I've also shot insulin, aprotinin, cytochrome c, and caffeine to check that the column is still good, but I haven't gotten conclusive results even with 50-500µg/mL solutions. But I find it hard to believe that after two 20µL injections of metabisulfite (sample and blank) containing 0.2µmol that the column is no longer any good. Any ideas of what may have happened to the peptide?

Well, I ended up having to get a new column. No one could figure out the reason the peptide would no longer elute. As soon as I ran with the new column, everything returned to normal. I guess we'll just have to avoid sodium metabisulfite on the Proteo column.

Thanks to everyone who considered the problem.

I had similar type of problem & in that case only changing new column can solved the problem.