HPLC-Protein and Peptide Separations

The Shared Instrumentation Core performs peptide and protein purification by reverse phase high performance liquid chromatography (HPLC) and can has access to facilities to separate proteins by more traditional gel filtration and ion exchange chromatography.

The Dionex U3000 HPLC is capable of using 0.3 mm, 2.1 mm, 1 mm, 4.6 mm diameter columns. The column diameter used depends on the sample size. Small columns give higher sensitivity and supply the sample in a small volume. However larger columns are needed for larger samples. When necessary, peptides from a digest are rerun on a different column or in different solvents to separate co-eluting peptides. Normally proteins and peptides are separated on C18 columns in mixtures of water and acetonitrile containing 0.1% trifluoroacetic acid.

In addition, we have performed some separations by ion exchange of small molecules using the HPLC systems more often used for reverse phase separations. We have purified a small acidic siderophore molecule and analyzed amounts of nucleotides.

Users may need to purchase a column for their projects. The instrument has only a UV detector. The Mass Spectrometry laboratory also uses HPLC in separating analytes and the mass spectrometer gives positive identifications of analytes.