Temperature vs retention

[Mattias]

I am currently doing method development in order to separate some degradation products from the main peak.

For this I try different gradients at different temperature (mobile phase 0.05% TFA in water/acetonitrile).

When I sit back and evaluate the runs, the retention time for some compounds is longer at the higher temperature (60°C) than at the lower (20°C). Isn't this physically impossible??

Does anyone have an explaination of what is going on!



BR/Mattias

[Cliff Mitchell]

I saw a paper by Berthod et. al. (J. Chromatogr. A, v1060, p205-214) examining the thermodynamics involved in chiral separations. They looked at 148 separations in reverse phase, normal phase, and a polar organic (nonaqueous) mobile phase mode, and found 19 cases where retention increased upon increasing the temperature. However, these separations were all in the polar organic mode (retention is dominated by hydrogen bonding & ion exchange).

ln k = -(dH/RT) + (dS/R) + ln (phase ratio)

In section 3.2 they discuss this, and in 3.4.2 this point out that these compounds had positve dH, and the entropy term (dS) was at least several times larger than the enthalpy contribution.

Basically, given the sign of the thermodynamic parameters for the partition/transfer process from the mobile phase to the stationary phase, it is possible for an increase in temperature to increase retention, it is just very uncommon. True this is a chiral study, but as it pertains to retention the theory is the same.

What kind of chromatography are you doing? Reverse phase C18?

[tom jupille]

Increase in retention with temperature is also quite common with reversed-phase LC of proteins. Thequalitative explanation is that more hydrophobic conformers of such proteins is favored at higher temperature.

[Mattias]

Thanks for your replies!

I am analysing peptides (approx 10 AA), and I am trying a Waters SunFire C18 column.

It looks as I can benefit form this "strange" behaviour, since the selectivity is different at the different temperatures!

[Broesen]

I am currently doing method development in order to separate some degradation products from the main peak.

For this I try different gradients at different temperature (mobile phase 0.05% TFA in water/acetonitrile).

When I sit back and evaluate the runs, the retention time for some compounds is longer at the higher temperature (60°C) than at the lower (20°C). Isn't this physically impossible??

Does anyone have an explaination of what is going on!



BR/Mattias

Hi Mattias.

High temperature is a hot topic at the moment. You will see that some times the selectivity can change with the temperature. If you tried temperature above 100C you will even see that water acts like an organic modifier.

Broesen

[Uwe Neue]

Analyte conformation is a neglected subject in HPLC, but such a phenomenon could be an explanation for your observations.

[unmgvar]

do your peptide posses a secondary structure?
at 60 degrees they would be denaturated and would be more a string then a complex like helix or beta sheets.
the change in the structure would increase the surface area that will react with the column. In an absorption type separation the increase of the organic surface area will result in an increase in the interaction between your compounds and the column.

[Mattias]

The peptide contain a sulphur bridge, which is still present in some of the degradation products.

I had another look at the data, and the degradation products that has a broken sulphur bridge has a "normal" temperature-retention time behaviour!

Can the temperature alone open a sulphur bridge?? I have not seen that in the stress studies. Maybe it is a reversible reaction, and the bridge is closed again at lower temperatures ?? (My knowledge in organic chemistry should be better...)

[Mark Tracy]

The disulfide bridge is opened by reduction and relinked by mild oxidation. 60 degrees is usually not enough to break the disulfide bond.

[unmgvar]

Do you have any other structures a part from the sulphur bridgein your protein?
in peptid geometry the sulfur bridge generally connect between 2 secondary structures to form a more complex and more thermodinamic robust geometry.

it is still possible that you do have other secondary structure that get denaturated.

from what you say concerning the degradation product that does remain in normal RT it seems logical to me that indeed you have a more complex structure in your product that changes at 60 degress. the degradation product containing only the structures related to the sulphur bridge is very stable and therefor does not change it's geometry due to the increment in temparature. your main compounds does

the sulphur bridge is exactelly one of the ways metabolisms use to protect their proteins from structural changes in different environment.

do you have sizeble degradation products which do not contain the sulphur bridge that also remain in the "normal" RT range at 60 degress? (they must be big enough to form helix or beta sheets).
if so then my 5 cents are totally off.