Chromatography Forum

Hi Forum,

I am getting set up to perform a series of (aqueous mobile phase) HPLC experiments at different temperatures. Right now I am thinking about the problem of keeping the pH buffered to a constant value even though the temperature varies.

I have acquired a (Waters) column temperature control module for this study. I am seeking to carry out all of my experiments at the same pH (4.4), even though the temperature varies (from RT up to about 70C), using DEPP buffer (N,N'-diethylpiperazine), which has a (relevant) pKa of 4.67.

Anyone have some very general advice on how to approach this problem?
I'm not expecting anyone to do any calculations for me, but to maybe help me go in the right direction or suggest pertinent literature or reliable web-based calculators.

Thanks,

David

In most LC experiments, the buffer capacity of a buffer is important, and this is a question of composition, and does not change with temperature or arganic composition.

I have serious doubts about the idea that your buffer would have a pKa of 4.67. Why don't you use an acetate buffer, which is the general practice in LC for a buffer with a pKa around 4.5?

Uwe,

I have to use a buffer in my experiments that does not react with my stationary phases. Recall that I am performing dynamic studies of strong adsorption, not doing separations. I am packing my own columns with metal oxide coated sands. Something like a carboxylate ion would consume all the surface sites, competing severely with the adsorbate/phenomenon that I wish to study. The buffer that I am using is one of a series of buffers that do not complex with metals be they in solution, in proteins, or on surfaces.

You were among the various experts that encouraged me to use buffers. However, I have to be selective about which buffers I use in these experiments.

These buffers are described in this paper: Yu, Q. Y.; Kandegedara, A.; Xu, Y. P.; Rorabacher, D. B., Avoiding interferences from Good's buffers: A contiguous series of noncomplexing tertiary amine buffers covering the entire range of pH 3-11. Analytical Biochemistry 1997, 253, (1), 50-56.

This highly cited article would address your question regarding the pKa value I quoted for TEEN.

Basic thermodynamics says that equilibrium constants, and hence pK values, for ionizable compounds depend on temperature. I am seeking to understand if a change in T shifts the weak acid/conjugate base concentrations. Of course it could not change the analytical concentration of the buffer.

David

You are right about the pKa of DEPP, and I have learned something. Thanks!

Me teaching you something? Now that is a real turn of the table! I'm sure the table will get back to its usual orientation in the very near future.

You can get an idea of temperature effects by playing with

http://www.bioinformatics.org/JaMBW/5/4/index.html