By Anonymous on Tuesday, June 29, 2004 - 11:47 am:

Can someone please explain to me the effects that pH will have on affinity chromatography (phenyl boronate stationary phase) and separation of glycated hemoglobins ?

Thanks in advance!!

By HW Mueller on Wednesday, June 30, 2004 - 02:50 am:

You may want to look at the other chains on buffers. Proteins are usually extremely influenced by pH: Changing their state of ionizatian, via pH, can starkly change their geometric structure as well (changing ionic and adsorptive interactions with the stat. phase). I am not familiar with your stat. phase, but presume that it´s also ionizeable, and thus needs to be controled. Incidentally, proteins can also be quite sensitive to changing ionic strength of the medium (mobile phase).

By HW Mueller on Wednesday, June 30, 2004 - 03:22 am:

Also, since an important interaction, here, may be that between the boronate and carbohydrate hydroxyls, the ionic state of the boronates should be decisive.

By Chris Pohl on Saturday, July 3, 2004 - 12:04 pm:

Two factors are involved in the case of the interaction of cis diols with a boronate stationary phase (from my point of view such a material is not properly described as an affinity phase but I know that a number of researchers have chosen to call non-biological selective chemistries "affinity chromatography" because they bear a vague similarity to true affinity chromatography). The first consideration is the pH necessary for the carbohydrate to react with the boronate species (the reaction product is often described in the literature as a complex but it is in fact a covalent compound). The pH needs to be at least seven in order for the two compounds to react at a reasonable rate. The reaction rate increases as the pH increases. The second consideration is the pH stability of the product. Above pH 11, the reaction product rapidly hydrolyzes, resulting in minimal retention. Thus, optimum conditions are a compromise between these two factors but also influenced by your intended mode of operation. pH nine tends to be a good compromise for both initial reaction and reasonable reaction product stability but you might get better retention in your capture step if you use the somewhat lower pH. Of course, assuming you're analytes can handle it, the higher your pH is the better when it comes to the release step unless you are using competitive displacement with another carbohydrate for release. In this case, operation of pH nine should suffice.