To buffer or not
Nov 29 2016 Read 660 Times
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One of the focus areas of this issue of Chromatography Today is on the analysis of large molecules. One approach that has gained significant interest to allow better classification of large molecules is the use of size exclusion chromatography. From a theoretical perspective it is one of the simplest separation mechanisms to understand as it relies on a physical segregation process instead of the traditional mechanisms using chemical interactions between analytes and stationary phases commonly seen in other modes of separation [1,2]. The differentiating mechanism is based strictly on the cross sectional area of the analytes and what pores of the stationary phase these molecules are able to access.
Aqueous size exclusion chromatography uses a non-interactive stationary phase coupled with an aqueous mobile phase. However, with the wide range of molecules being analysed by this powerful technique, there are occasions when secondary interactions between the stationary phase and the analyte occur. These undesirable interactions alter elution times and also result in peak shape deformation away from a true Gaussian profile. Antibody drug conjugates (ADC’s) are particularly prone to these secondary interactions which is not particularly surprising since most secondary interactions are polar in nature and ADC’s as a class of compounds are in fact known for their high degree of polarity [3,4]. The ADC’s high degree of polarity  is one of their attributes that contributes to their retention and ideal chromatographic behaviour on diol stationary phases.
There are some obvious solutions to reducing the amount of secondary interactions between the analyte and the stationary phase in SEC. One approach is to use buffers while another approach is the addition of an organic additive to the mobile phase. In both situations, the desired result is for the compound to not chemically interact with the surface of the column and instead elutes based on the pore volume of the stationary phase that it is able to traverse through. So the obvious question what effect does using buffers and organic solvents have on the analysis of proteins using SEC?
To understand the unrealised benefit of using this approach let’s examine the structural properties of a protein  as well as how proteins interact with other molecules. Proteins typically have three definable levels of structure: the primary structure relates to the arrangement of the amino acids or the amino acid sequence, the secondary structure is due to regularly repeating local structures stabilised by hydrogen bonds, and finally a tertiary structure which is obtained by nonlocal interactions, most commonly the formation of a hydrophobic core, but also through salt bridges, hydrogen bonds, disulphide bonds, and even post-translational modifications.
In This Issue... Articles - Industrial Applications of Offline and Online Comprehensive Gas Chromatography - Improving Workflow in Group-Type GC×GC Analysis of Petrochemicals - Identifi ca...
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