Chromatography Forum

Hi all I ma having trouble with my amino acid method with active sites. The method is known to be quite sensitive to active sites. I am noticing a drop and even complete disappearance of the most polar analytes specifically ethyl chloroformate derivitized lysine, histidine, and glutamine. I replaced the septa and liner and tried washing the column with my solvent pump kit (MeCl2 followed by 20%water in MeOH). The separation is fine and the nonpolar amino acids are fine but the polar ones are vanishing. Column is a DB-5MS injection is 10:1 split He with a Agilent 4647 liner.

Would injecting a silylating reagent help?

First, determine if the column is the problem. Install temporarily a new column and see if the problem goes away.

If it does, then try to fix the old column, or throw it away.

best wishes,

Rod