Choice of Phase Systems in MD-LC

Separation methods exploiting different physical properties of proteins have been combined with varying degrees of success. The ultimate goal is a rapid separation strategy that can be coupled with detection methods, such as MS, to provide 

Ideally, components that are not separated in the first separation step are resolved in the second. Peak capacity is the number of individual components that can be resolved by a separation method. A mathematical model shows that if the MD separations are orthogonal, then the total peak capacity is the product of the individual peak capacities of each dimension [14], Load capacity is defined as the maximum amount of material that can be run in a separation while maintaining chromatographic resolution. MD separations can be designed to significantly increase the load capacity in a first dimension to achieve enrichment of low-abundance or trace components in a peptide mixture, while the necessary peak capacity may be obtained in the second separation dimension [15]. 

The primary criteria for the choice of a separation phase system are selectivity and orthogonality, mass loadability, and biocompatibility (in case of quantification). 

As a rule of thumb, the first dimension should possess a high mass loadability combined with sufficient selectivity and maintenance of bioactivity. Ion exchange chromatography (lEC) is therefore the method of choice offering charge selectivity. 

In principle, there are two options in lEC, to employ either a cation or an anion exchanger, which in turn influences the pH working range. Note that either cationic or anionic species are resolved, i.e., only a limited number of species from the whole spectrum. The I EC columns are operated via salt gradients with increasing ionic strength. Consequently the salt load must be removed before the fractions are transferred to the second dimension column. 

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