2D-HPLC is becoming more and more popular in labs that have to perform analysis of complex mixtures of proteins.
When coupled to a mass detector, high levels of sensitivity can be achieved.
This technique generally require three columns :
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A strong Cation Exchanger for the separation of protein groups
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An apolar (usually C18) column for trapping
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An apolar column for the final sample analysis
The sample is loaded onto the initial SCX column and the different protein fractions are eluted by a buffer with successive increased concentration.
The second (short length) apolar column traps and concentrates the proteins all allows for the changing of solvent and a discarding of salts and contaminants. Sample analysis of all fractions are performed on the third apolar column. This column is typically a C18 or C4 120 Å or 300 Å support.
Productivity can be increased by a modification of the third phase of this process. By utilising a 10 way/2 position valve instead of the standard 6 way/2 position unit a second HPLC column can be installed into the system. Protein fractions can then be loaded alternatively onto each column.
