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VYDAC Bonded Phase: Effect of Pore-Size, Buffer, pH and Column Chemistry on Peptide Purification

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Summer, 1998 excerpt

Effect of Pore-Size, Buffer, pH and Column Chemistry on Peptide Purification

Different C18 Bonding Chemistry Provide Selectivity Alternatives.

Diversity is an innate characteristic of peptide structures. Even closely related peptides, for example target product and contaminating side products from a synthetic process, will typically have chemical differences at multiple sites consisting of various hydrophobic, polar, and charged acidic or basic constituents.

This remarkable diversity is what allows peptides to exhibit a wide range of biological activities. It also provides chromatographers with the ability to change selectivity and control resolution in reversed-phase separations to a much greater extent than is normally possible with smaller, simpler molecules. Providing tools and understanding to make this possible is one of Vydac's missions.

The chromatograms of Figure 1 show separations of the same synthetic peptide mixture on two different reversed-phase columns under identical conditions of flow, temperature, and organic solvent gradient. The separations differ in the pore-size of the packing and the pH and buffer ions in the aqueous mobile-phase component. They represent extremes of a series of resolution trials aimed at finding the best conditions for purification of the major component, a partially purified cyclic peptide with a C-terminal acyl hydrazide. Each chromatogram reveals one large peak (inset), the target product, surrounded by several much smaller contaminating peaks. The vertical axis in the region containing the major peak has been expanded to show the resolution in detail.

Figure 1. Peptide purification under typical conditions (A) and after optimization (B). The region around the main peak has been expanded to show detail. (Inset shows complete chromatogram for 218TP54.)


Chromatogram A of Figure 1 was the first attempt at reversed-phase separation using a 90Å pore-size C18 column (Vydac 201HS54) with the mobile phase most commonly used for peptide separations, TFA/ACN. Chromatogram B, a separation at pH 6.0 in triethylamine acetate (TEAA) buffer on a 300Å pore-size C18 (Vydac 218TP54), represents perhaps the best option resulting from the series of trials shown in Figure 2.

Figure 2. Comparison of synthetic peptide separation on three columns under five different buffer conditions.
All columns: 5µm, 4.6mmID x 250mmL. Temperature: Ambient. Flow rate: 1.0 mL/min. Buffers: 0.1% TFA (v/v). 10mM TEA Phosphate, pH 2.0. 10mM NaH2PO4, pH 2.0. 10mM TEA acetate, pH 6.0. 10mM NaH2PO4, pH 6.5. Mobile phase A: Buffer as indicated. Mobile phase B: Buffer as indicated (except 0.09% for TFA), 90% ACN (except 80% ACN for phosphate buffers). Gradient: Initial 5% B to 2 minutes. Then 20 minute linear to 45% B. Then 2 minutes to 100% B. Hold 2 minutes. Return to 5% B in 1 minute. Detection: 215 nm. Sample: Partially purified cyclic peptide with C-terminal acyl hydrazide.

Several conclusions can be drawn from inspection of the complete series in Figure 2. First, it is clear from comparisons under any of the mobile phase conditions tried that the 300Å wide-pore reversed phases produce narrower peaks and improved resolution when compared to the 90Å adsorbent. Peptides of significant size, or rigidity as in the case of a cyclic peptide, will be at least partially excluded from the pores of the 90Å material. In addition, narrow pores restrict diffusion. Both effects contribute to peak broadening. Thus, for peptide separations in general, 300Å wide-pore silicas should be the adsorbents of choice.

Effects of buffers and pH are dramatic. In general, separation at mildly acidic pH (6.0 or 6.5) appears to produce better peak shape and resolution, or at least a greater number of resolved components, than separation at highly acidic pH. This observation is significant in view of the fact that highly acid conditions such as 0.1% TFA tend to be the norm for reversed-phase peptide chromatography. The results shown here suggest that pH 6.0 or 6.5 separations are more likely to separate difficult to resolve components. However, it should be noted that these results are specific to the particular peptide sample used. The conclusions may or may not apply to other samples.

Significant selectivity differences are seen between the two chemically distinct 300Å reversed phases. Vydac 218TP is a polymeric C18 produced with multifunctional silanes, while 238TP is a monomeric C18 produced with monofunctional silanes. In specific situations, one or the other of these adsorbents may produce better resolution between critical components. In some instances, it may be desirable to run sequential separations on different adsorbents or with different buffers to eliminate specific problem contaminants.

As shown here, running a series of trial separations can help greatly in choosing optimal conditions for a specific peptide purification. For process development, where extensive method workup can be justified, use of a resolution mixture will also yield additional information about the sample, assist in optimizing conditions, and provide a means for process validation.

Data for this article courtesy of

Tim Malaney
Telios Pharmaceuticals, Inc.
San Diego, CA

Ordering Information:

Vydac Cat. # Description
201SP54 Column, Octadecyl (C18), Monomeric, 5µm, 90Å, 4.6mm ID x 250mm L 
218TP54 Column, Octadecyl (C18), Polymeric, 5µm, 300Å, 4.6mm ID x 250mm L
238TP54 Column, Octadecyl (C18), Monomeric, 5µm, 300Å, 4.6mm ID x 250mm L

Other analytical and preparative column dimensions available upon request.

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