Fifteen years ago, the introduction of synthetic, high purity silica with wide pores made the chromatographic separation of peptides possible and practical. The separation selectivity of peptides on silica-based reversed-phase columns is not only determined by the nature of silica, but also by the type of silanes and bonding chemistry used. This provides us an important tool to design columns with different selectivity to suit different separation needs. In this report, we demonstrate that different silanes applied on the same silica have different selectivity for peptide separations. The comparison results show that these reversed-phase columns can be used complementally for peptides which are difficult to separate.
Vydac TP is a high-purity 300Å pore-size silica produced from purified organic silicates.
Historically, protein/peptide reversed-phase adsorbents produced from TP silica have had "polymeric" bonded phases synthesized from polyfunctional silanes which produce crosslinking of the hydrophobic phase on the silica surface. This produces columns with long lifetimes and no measurable stationary phase leaching. Vydac 218TP is a C18 reversed-phase adsorbent produced in this way.
Bonding Chemistries
More recently, we have bonded a "monomeric" C18 ligand on the same silica which is later exhaustively end-capped. The resulting adsorbent is designated Vydac 238TP. Figure 1 shows a comparison of peptide separations on the polymeric and monomeric bonded phases. Note the
Figure 2 shows that changing the concentration of TFA modifier in the mobile phase is another way to change selectivity. For reproducible runs it is important that TFA concentration be clearly specified (v/v or w/v) and carefully controlled.
It can be seen from the chromatograms in Figures 1 and 2 that under identical operating conditions 218TP and 238TP exhibit comparable retention of peptides, but subtle differences in selectivity. This can help in separating peptides that are not resolved or incompletely resolved if only one reversed-phase column type is tried.
Figure 1. Synthetic peptides on two 300Å C18 silica reversed-phase columns. Both columns were 4.6 mm ID x 250 mm L. Chromatographic conditions were identical. Conditions: 1.0 mL/min, absorbance at 220 nm, gradient from 10% to 40% ACN with 0.1% TFA (w/v) over 30 minutes. Sample: Mixture of standard peptides, listed with peak identification: 1>GY, 2>VYV, 3> neurotensin fragment 1-8 (pELYENKPR), 4>acRGGGGLGLGK-NH2, 5>RGAGGLGLGK-NH2, 6>acRGAGGLGLGK-NH2, 7>acRGVGGLGLGK-NH2, 8>oxytocin (CYIQNCPLG-NH2), 9>met enkephalin (YGGFM), 10>bradykinin (RPPGFSPFR), 11>acRGVVGLGLGK-NH2, 12>neurotensin fragment 8-13 (RRPYIL), 13>angiotensin II (DRVYIHPF), 14>leu enkephalin (YGGFL), 15>neurotensin (pELYENKPRRPYIL), and 16>angiotensin I (DRVYIHPFHL).
Figure 2. Effects of reversed-phase column type and TFA concentration. The bottom trace is a complete chromatogram. The upper traces show the region from 15 to 85 minutes, in which virtually all tryptic peptides emerge, for four individual runs with the vertical scale expanded to facilitate comparison. Samples identical for all runs.Conditions: 1.0 mL/min, absorbance at 215 nm, TFA concentration (w/v) as indicated, gradient from 0% to 50% ACN over 100 minutes. Both columns were 4.6mm ID x 250mm L. Sample: Tryptic digest of apotransferrin.
| Vydac Cat. # | Description |
|---|---|
| 238TP54 | Column, Octadecyl (C18), Monomeric, 5µm, 300Å, 4.6mm ID x 250mm L |
| 218TP54 | Column, Octadecyl (C18), Polymeric, 5µm, 300Å, 4.6mm ID x 250mm L |
Larger columns are also available.