Peptide fractionation is extremely important in proteomics approaches. Full proteome characterization is desired from complex organisms, and with growing interest in post-translational modifications an extended protein sequence coverage is required. Peptide fractionation techniques have the great challenge of feeding current mass spectrometers in a way in which these issues are met. Peptide fractionation can be divided into three simple components: the column characteristics; the mobile phase; and peptide properties (charge, polarity, hydrophobicity and size). The current challenges are in the combination of these three components to allow comprehensive proteomics studies to be improved.