Mixed‐mode hydrophilic interaction/cation‐exchange chromatography: Separation of complex mixtures of peptides of varying charge and hydrophobicity

Journal of Separation Science - Tập 31 Số 9 - Trang 1573-1584 - 2008
Colin T. Mant1, Robert S. Hodges1
1Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, CO, USA. Fax: +1-303-724-3249

Tóm tắt

AbstractMixed‐mode hydrophilic interaction/cation‐exchange chromatography (HILIC/CEX) was applied to the separation of two mixtures of synthetic peptide standards: (i) a 27‐peptide mixture containing three groups of peptides (each group containing nine peptides of the same net charge of +1, +2 or +3), where the hydrophilicity/hydrophobicity of adjacent peptides within the groups varied only subtly (generally by only a single carbon atom); and (ii) peptide pairs with the same composition but different sequences, where the sole difference between the peptides was the position of a single amino acid substitution. HILIC/CEX is essentially CEX chromatography in the presence of high levels of organic modifier (generally ACN). The present study demonstrated the dramatic effect of increasing ACN concentration (optimum levels of 60–80%, depending on the application) on the separation of both mixtures of peptides. The greater the charge on the peptides, the better the separation achievable by HILIC/CEX. In addition, HILIC/CEX separation of both the peptide mixtures used in the present study was shown to be superior to that of the more commonly applied RP‐HPLC mode. Our results highlight again the efficacy of HILIC/CEX as a peptide separation mode in its own right as well as an excellent complement to RP‐HPLC.

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Journal of Separation Science

Tập 31 Số 9

1573-1584

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