Peptide Separation in Normal Phase Liquid Chromatography

A new method is established for separating peptides in normal phase liquid chromatography using TSK gel Amide-80, carbamoyl groups bonded to a silica gel matrix, and an acetonitrile-water solution containing 0.1% trifluoroacetic acid. Peptide retention time increased with acetonitrile concentration in the initial eluent. Hydrophilic peptides with no retention in a reversed phase column were retained and separated in the present method. Separation selectivities in the present and reversed phase methods differed significantly. Two-dimensional separation of protein digest using reversed and normal phases was conducted, taking advantage of the differences in selectivities. All peptides obtained from the digest could be separated completely. The present method is useful for separating peptide mixtures in conjunction with reversed phase liquid chromatography. Peptide recovery from the Amide-80 column exceeded 80%, as with the reversed phase column, and repeatability and reproducibility were satisfactory.