The Chromatography Forum

of Delaware Valley

Abstract:  

This lecture divides into two topics:

HILIC: Increasing the concentration of the salt in the mobile phase increases the mass of the immobilized aqueous layer. There is a widespread belief that this results in greater retention of polar solutes. The lack of evidence for this effect has led us to examine it through controlled experiments with neutral solutes. Preliminary results suggest that the effect depends on the selection of the salt involved, which can tune up or tune down hydrogen bonding.

Top-Down Proteomics: Lack of suitable separations is a major bottleneck in this field.  We are addressing it in two ways:

    a) SEC: Proteins < 40 kDa suppress the detection of proteins > 40 kDa. SEC separates these two sets of proteins, making it possible to use top-down MS to identify larger proteins and their minor variants with post-translational modifications such as phosphorylation and acetylation.

    b) RPC is a high-resolution mode but denatures proteins. This makes analysis by MS more complicated. We have introduced a new line of materials for hydrophobic interaction chromatography (HIC) that permit the separation of proteins with high resolution and with native structure intact. Elution can be performed using concentrations of ammonium acetate that are compatible with direct MS analysis. This will prospectively eliminate the necessity of digestion and peptide mapping for analysis of antibodies and antibody-drug conjugates.