HPLC

The Agilent 1100 Series Purification System is available for analysis and purification of proteins and peptides.

Theory

• Reverse-phase columns separate peptides on the basis of hydrophobicity.
• Reverse-phase is the principle type of column used in most single dimension LC and LC-MS/MS experiments.
• Compounds stick to bind to the hydrophobic stationary phase in HPLC columns in high aqueous mobile phase and are eluted from the column with increasing organic mobile phase.
• In the aqueous phase peptides bind to the hydrophobic column material through a hydrophobic interaction then are eluted according to relative hydrophobicity as the organic eluting buffer begins to disrupt these interactions.
• Peptides are separated by running a linear gradient of the organic solvent.
• The more hydrophobic the peptide the higher the organic concentration required to elute the peptide from the reversed phase column.

Typical Experiments and Procedures

• Peptide mapping on RP-HPLC with collection of peptide fractions.
• QC analysis of purified peptides and proteins.
• Purification of functionally active proteins and peptides. A full range of chromatography techniques are available including gel filtration, ion exchange, reversed phase and affinity.

Sample Information and Preparation Required

• Sample origin and type is helpful deciding appropriate HPLC separation conditions.
• Knowledge of sample buffer is important because not all buffers are compatible with HPLC eg presence of detergents.
• Sample must be free of any particulates or other insoluble material and should be filtered or centrifuged prior to injection.