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Peptides are used in basic research as well as in biopharmaceutical development. It is common to use synthetic peptides rather than isolating natural products. Such synthetic products may not be sufficiently homogeneous for use in experiments requiring bioactivity and specificity. Isolation and purification, therefore, become essential steps in a peptide-synthesis facility. We have examined the factors that influence purification protocols. This includes selection of a column, operating parameters based on the properties of the peptides, and the intended use of the product. A panel of synthetic peptides covering a range of properties including size, hydrophobicity, and isoelectric point is used for these evaluations. These test samples are used to measure the importance of pore size and particle size. Under these controlled conditions, there is little difference between 130 Å and 300 Å up to 40 residues. More resolution was observed with 5-μm particles than with 10 μ. The uses of the purified peptides may constrain the mobile-phase constituents. The suitability of biocompatible mobile phases, including acetic acid in place of trifluoroacetic acid and ethanol instead of acetonitrile affected the selectivity of the separation. In some comparisons, selectivity effects could be related to the peptide sequence, while other changes are still open to interpretation. An additional set of experiments has been performed investigating conditions for classes of peptides that have extreme or special properties. We show the impact of elevated temperature as well as the use of high pH and/or alternative solvents for peptides that are very difficult to dissolve in common chromatographic solvents. These experimental observations are combined into a suggested protocol for developing an isolation method.