Chromatography Forum

The idea is that, at ambient temperature, the peptide binds the reversed-phase column as different species with varying degrees of denaturation. The peptide elutes as a broad or non-Gaussian peak from the different forms. When temperature is increased the peptide becomes completely denatured. There is now one species, and it elutes as a sharper, properly shaped peak.

My question:
I understand temperature can denature tertiary (and certainly quaternary) forms. Is the secondary structure (denaturation) important for sharpening peaks?

I am working with 3-5,000 MW peptides. These probably have minimal tertiary structure, mostly secondary. So, how important is the temperature for these analytes? Is the secondary strucure preserved on reversed-phase HPLC?

If not, how much of a difference will temperature make, and what is the temperature range at which a difference in peak shape will be observed?

For an academic guess at this it may be helpful to know the answer to the following questions: Did you already show that you peptide(s) have an improved peak shape at higher temp.? Does this happen over an extended or narrow temp. range? Does the increase in temp. increase or decrease the retention time?

If not, how much of a difference will temperature make, and what is the temperature range at which a difference in peak shape will be observed?

"The devil is in the details". Remember that your stationary phase can only interact with the outside of your peptide. Any change in the molecule's shape may by reflected in the chromatographic performance. Even a "minimal" change in conformation (tertiary structure) may make a significant difference.

You only need to worry about denaturing if you want to collect and use the protein. Otherwise, if you only want to analyze it, do what you need to do to get a good peak shape.

In general, higher temperature should improve peak shape. Check out applications on the column manufacturers' web sites, and note the temperature. I know Agilent published sevaral notes on antibody separations (about 100 kD size) using the Poroshell (fused core) material. The authors indicated that high temperature (70 C) was needed for best peak shape. They also using 0.1% TFA and 0.3% PEG 300 in the mobile phase. I was unable to directly reproduce this work, but also was unable to exactly duplicate their conditions. But I believe the idea is worth considering.