HPLC-protein and peptide separations

HPLC-protein and peptide separations

The Shared Instrumentation Core performs peptide and protein purification by reverse phase high performance liquid chromatography and can also separate proteins by more traditional gel filtration and ion exchange chromatography.

For reverse phase separations, there are instruments suitable for  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.

The Facility also has an FPLC for ion exchange or gel filtration separations of proteins. Detection is UV absorbance at 280 or 254 nm. Our instrument has a  fraction collector.

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.