Selectivity of Peptides on Amide Embedded Polar Group Phases Versus Alkyl Phases

By: Hillel Brandes and Patrick Myers, Reporter EU Volume 20

Hillel Brandes and Patrick Myers


It has been known that small molecule analytes with hydrogen bond donor moieties display enhanced retention on embeddedpolar group (EPG) phases(1, 2, 3). However, no such systematic study has been done with peptide analytes, until this report. Synthetic peptide sets, in which phenylalanyl residues were substituted with tyrosyl residues, were used to inspect relative differences in retention as a function of the degree of substitution. Indeed, substitution with the phenolic hydroxyl of tyrosine (versus a single hydrogen of phenylalanine) does confer enhanced retention of the peptide on the EPG amide phase. This is consistent with results using small molecules.

back to top


Ever since EPG phases were first reported (4), they have remained popular for chromatographing polar analytes. While these phases do retain compounds based on hydrophobicity, polar interactions can significantly contribute to retention as well. Deciphering the molecular interactions that confer particular retention on EPG phases has been of considerable interest. Various studies with small molecule analytes have indicated that amide EPG phases display particular retention for compounds that may function as hydrogen bond donors (1, 2, 3). While we have previously noted different selectivities of peptides on alkyl versus EPG phases, no systematic study has previously been performed to elucidate what molecular interactions may be responsible for the altered retention. In this article we report the first such investigation by inspecting the relative differences in retention of two sets of peptides, in which phenylalanyl residues of one set are substituted with tyrosyl residues in the other set. One of the more clear cases with small molecules, which show enhanced retention on amide phases, are phenols. Therefore, this comparison of phenylalanyl and tyrosyl residues was a logical first case to inspect possible molecular interactions that contribute to alternate selectivity of peptides on EPG phases.

back to top

Methods and Results

Peptides obtained from Sigma-Genosys; chromatographic conditions indicated with figures.

Peptide set I (in order of elution): 1. FGGAGGAGGG-amide 2. FGGFGGAGGG-amide 3. FGGFGGFGGG-amide 4. FGGFGGFGGF-amide

Peptide set II (in order of elution): 1. YGGAGGAGGG-amide 2. YGGYGGAGGG-amide 3. YGGYGGYGGG-amide 4. YGGYGGYGGY-amide

Figure 1 shows the retention of the peptide set I set on Ascentis C18 and Ascentis RP-Amide. For this peptide set, retention is conferred primarily by hydrophobicity. Thus the lower hydrophobic retention of the amide phase (as compared to C18) is demonstrated by the lower retention of the peptides. Figure 2 shows the retention of peptide set II on the two phases. Absolute retention differences of each peptide set on the two phases are not pertinent to this study but arise from the larger hydrophobic retention index of phenylalanine versus tyrosine (5, 6). The clear difference in comparing the retention of each peptide set on the two phases, is that in the case of peptide set II, enhanced retention of the tyrosyl-substituted peptides occurs in an incremental manner with each additional substitution. That is, the difference in retention of each peptide (of set II) on the amide versus C18 increases as the number of tyrosyl residues increases. Thus, the phenolic hydroxyl of the tyrosyl side chain is conferring enhanced retention in a manner consistent with what has been previously observed with small molecules. If retention on the amide phase were strictly hydrophobic, the comparative elution patterns versus C18 for peptide set II would look like that of peptide set I (less retention of all peptides of set II on the amide phase versus the C18 phase).

Figure 1. Phenylalanyl Peptides (Peptide Set I) on Alkyl and Polar-Embedded Phases
565305-U 581304-U

Figure 2. Tyrosyl Peptides (Peptide Set II) on Alkyl and Polar-Embedded Phases
565305-U 581304-U

back to top


We have shown conclusively for the first time, that hydrogenbonding donator moieties of peptides confer enhanced retention on amide EPG phases in a manner consistent with what is observed with small molecules. This is likely to occur with other EPG phases that contain a carbonyl group.

We intend to expand this study with other possible hydrogenbond donors and acceptors of peptide side chains to further investigate which peptide functional groups may contribute to altered selectivity on EPG phases versus alkyl phases.

back to top




  1. Supelco poster. Separation Problem Solving Through Selectivity -Comparison of a New Stable Polar Embedded Phase with a New ODS Phase; T404130
  2. Supelco poster. Designed HPLC Selectivity Enhancement; T405015
  3. Supelco poster. Retention Mechanisms In Reversed-Phase Liquid Chromatography: Embedded Polar Group Stationary Phases. T405086
  4. Ascah, T.L. & Feibush, B. 1990. J Chrom 506: 357-369
  5. Guo et al. 1986. J Chrom. 359: 499-517
  6. Sakamoto Y. et al. 1988. J Chrom 442: 69-79

back to top

Related Links