Chromatography Forum

Hi all!

I need your expert advice on a method i am developing in the lab. I am trying to develop a method to separate a ten residue peptide that is hydrophilic on a TSK Gel amide 80 column. I am using 0.1%TFA+100%Acetonitrile as solvent A and 0.1%TFA as solvent B. My problem is that i do not get sharp peaks. The ID of the column is 1mm and flow rate 30uL/min.Elution is done with increasing water.
Can you advice me on this method or tell me if i am doing things correctly or an alternate method.
I am also trying HILIC/CEC using Polyhydroxyethyl A column with TEAP and NaClo4 but the retention times keep changing.

Thanks!

Roy

The first question is "what, exactly, do you mean by not getting sharp peaks?" (seeing a chromatogram would help!).

Second question: "have you ever gotten sharp peaks from this column (e.g., running standards)?".

Third question: "In what are you dissolving your sample?".

Fourth question: "What is your injection volume?".

One of the concerns with small columns is extra-column volume; another with any gradient is peak distortion from injecting a sample dissolved in a "strong" solvent (which, if you are really doing normal phase, would be highly aqueous).