Chromatography Forum

Hi

I would like to know the behavior of a “compound “ in reverse phase with respect to its molecular weight…lets say there is a sample of molecular weight 5Kda, 20Kda and 150Kda..What will be difference of their behavior in Reverse phase??

Regards

thanks

I would tend to expect no change with respect to partition coefficients ("reversed phase" separation mechanism is mostly keyed to this), but you might see some size exclusion effects. You are talking about a 30x increase in mass & nearly the same in size. I suspect you'll do better with a GPC column than a traditional RP one.

The changes in retention with mobile phase composition becomes very steep with high-molecular-weight compounds. You will be able to use gradients only. An attempt to do isocratic chromatography will be futile for the largest molecular weight, and difficult for the smallest molecular weight.

hi
thanks for replies...

let me rephrase
if one subject the above 3 molecules to RP chromatography (-gradient elution) will it be difficult to separate closely related impurities (or species with very slight variation ) as the molecular weight increases?? if so why??

I would suggest that you pick up a copy of "Practical HPLC Method Development" by Snyder, Glajch, and Kirkland. That has a very good discussion of the effects of molecular weight on reversed-phase. The "S-value" (slope of log(k') versus fraction strong solvent) is molecular weight dependent, increasing in rough proportion to the square root of the molecular weight. By itself, that has little bearing on the ability to separate close compounds.

In some cases such as large peptides or proteins, for example, very small differences in sequence can result in big differences in conformation and very easy separation.