In an IEX HPLC of a protein at pH 6.6 I see a small increase in RT when I increase temp. I don't understand.

You really haven't given us enough information to help you. You haven't even told us the type of column. I would guess that the change in retention is due to the temperature change changing the pH of your buffer.

Lets say you are performing a cation exchange separation of a MAb at pH 6.6 and the pI of your MAb is 7.5. At pH 6.6 your MAb will have a net positive charge. Many organic buffers show a small change in pH when you change the temperature. Lets say you are using ACES and the pH decreases 0.02pH units/C. If you increase the temp 10C you will shift the buffer pH 0.2 units lower and your MAb will have a larger net positive charge and an increased retention.

Anyway, just look up how your buffer pH changes with temp.

In ion exchange you´re working with thermodynamically characterized exchange processes on your matrix. This means the retention of your solute(protein) is described by its free enthalpy of adsorption and desorption:
lnk = (DeltaG(adsorption)/RT) + ln(V(stationary)/V(mobile).
Therefore your retention is directly temperature dependent.

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To Alexander,
Try this link:
http://www.rheodyne.com/lit/docs/2320157C.pdf

Anon. Aug. 29: Your formular indicates a decrease of RT with an increase of temp. This holds for most, smaller, molecules. Proteins often do the opposite, as in the original question. This is said to be an entropic effect. (Would have to look up my notes to explain more thoroughly)(We could well stand a thermodynamics expert in the group)

Hi Hans,

Do you have data that shows small molecules and proteins responding in opposite fashion to a change in temperature in the same buffer system? If so could you e-mail me.

Best Regards,

Tom

Actually, there are many possible explanations for changes in retention time relative to temperature when it comes to protein separations. In the first place, temperature effects the ionization of the buffer, the ionization of the protein (carboxylic acid functionalities generally reach a maximum ionization near room temperature whereas ionization of amine functionalities increase progressively with increasing temperature, so one can expect pI of the protein to be temperature dependent) and in the case of weak ion exchange phases it also effects the extent of ionization of the stationary phase. In addition, even if none of the above are responsible for the phenomenon, it is generally true that ions with valency > 1 exhibit increases in retention with increasing temperature. This is true whether the ion is as small as sulfate or as big as albumin. So, I would generally expect to see increased retention with elevation of temperature unless some of the previous mentioned parameters are sufficient to offset this general phenomenon.

Sorry, my mind had switched to RP by the time the reply was decided. Tom, you probably don´t need them, but here are some refs on RP: Hearn in Janson and Ryden, Protein Purification, VCH, p. 199 (probably any other review by Hearn will do); Ooms, LC GC Int., 9, 574(1996); Snyder Glajch Kirkland, Practical HPLC Method development, Wiley, 1988. (Own examples are limited to small molecules whose RT decreased with increase in temp.)

Back to ion exchange: I have little experience in this area, but, Chris, what is the thermodynamic background behind the "rule" you mention regarding ions with charge > 1? It is certainly not enthalpy, must be an entropic effect. The other factors you mention tend to give a decrease of retention with increase of temp. So maybe entropy is still the most likely explanation for the original observation?

Indeed, I believe you're correct that the effect is related to changes in entropy. The structure of water within an ion exchange material is quite a bit more disordered than is the case the bulk water and hence the entropy term can be significant in terms of selectivity. As the temperature increases the structure of the bulk water loses much of its order due to substantial decreases in the hydrogen bonding interaction between water molecules whereas increases in temperature have a significantly smaller effect on the are ready disordered water structure in the stationary phase. So I guess you could attribute it to the improved ability of polyvalent species to enter the disordered environment of the stationary phase because of the decrease in the relative entropy of the two phases, allowing the electrostatic term to play a more significant role in retention.

Question: with increase temperature, could you also be changing the shape of the protein? Wouldn't this affect RT as different functional and stearic groups are accessable to the stationary phase?

original Anon, what sort of temperature increase are you refering to? 1 Kelvin or 30?