Chromatography Forum

I work with relatively hydrophobic, basic natural peptides between 5-9000 Da. Generally, 35-45% MeCN (0.1% TFA) is required for elution from C4 columns.

I have read that elevated column temperatures generally improve recovery and peak shape.

If I understand correctly, a reason elevated temp improves peak shape is by DENATURING the ligand, so that it separates as a single species (as opposed to various *partially* denatured forms which presumably behave as multiple species).

I'm somewhat confused about this, as I also understand that denatured peptides/protein can lead to irreversible binding to the RP column (=poor recovery).


Can anyone clear this up?

You need to differentiate between "denatured" forms of peptides and proteins. The opening of the structure of peptides will facilitate a uniform elution and therefore results in narrow peak shapes. In modern times, there should be no elution problem with peptides in RP. Proteins are much more complicated beasts with disulfide bonds all over the place etc. In this case, "denaturing" may result in crosslinking and lack of elution of the crosslinked stuff from the column.

What pore size are you using? Usually 12nm pore size will do the trick
for these peptides. But, sometimes you may benefit from 30nm.

Also, a lot of work has gone in to making HPLC columns for peptides and proteins.
The final product has been optimzed to allow for improved peak shape and recovery.

Some column manufacturers are better at this than others.