Chromatography Forum

can HPLC column separate peptide with L-amino acid and D-amino acid

thanks and regards

Chirobiotic T and Chirobiotic TAG are the best columns for separating chiral amino acids.

If the other aminoacids in the peptide are also chiral you should have diastereomeric interactions if one amino acid is replaced with its optical antipode. Theoretically you should be able to separate diastereomers with an achiral column.
Not having been satisfied with only SEC of proteins I started reading into reverse phase LC, unfortunatly never really got going (failed with isocratic methods). I mention this, because it seem to me that people should know this is a challenging area.

Hans -

Do column manufacturers claim that RP separation of proteins can be done by isocratic elution?
If they are, they shouldn't. Gradient elution is required for RP of proteins.

There were no suggestions, there were examples of peptides and (now I see it more clearly) small proteins being chromatographed with isocratic RP. Well, I still don´t see clearly enough where the molecular weight limit is, but the Mab (~150000) couldn´t be tricked to behave as I wanted. (Different pH, organics, chaotropic additives, . . . none prevented the "all or nothing" behavior).

That is certainly possible. We run a method that separates L-Arg desmopressin and D-Argdesmopressin with a resolution of >2.0. Standard column, standard mobile phase.

the sample is peptide,only one chiral amino acid

HI mattias:

do you mean C18 column,0.1%TFA in water as mobile phase A, and 0.085% TFA in ACN? if this mobile phase does not work, do you have some other suggestions?

thanks and regards

Desmopressin only contains ten amino acids - I guess it will be harder the larger molecule you have.

Mobile phase has almost no effect on the separation. Run different columns and see which one that gives you most separation (Zorbax SB-CN or Sunfire C18 works in my case). It will probably have to be isocratic elution.