Slow-gradient, sample-displacement chromatography

A universal, one-step method for the purification of large quantities of peptides

High performance liquid chromatography (HPLC) is one of the premier chromatographic techniques used world-wide for the purification of a wide variety of chemical compounds. It is essential for the isolation of new natural products, often used in the purification of synthetic molecules, ubiquitous in the peptide and protein synthesis lab, and indispensable for analytical chemistry. 

Figure 1. Schematic 
representation of molecule
elution profiles at different
loading levels: a) full peak
resolution, b) loss of 
resolution due to column
overload, c) displacement
chromatography

For the most part, peak resolution is required to achieve purification by HPLC, regardless of which mode (e.g. normal phase, reversed phase) is being used.  This means the compound of interest has to be clearly separated from any impurities, as depicted schematically in Figure 1a.  This limits the quantities of compound that can be loaded onto the column, before resolution is lost due to sample overload and peak overlap (see Figure 1b).  Purification of large quantities of compound therefore requires either multiple, repetitive runs, or the use of large (and expensive) prep columns and large quantities of solvent.

Recently, researchers from the Brimble group at The University of Auckland, New Zealand, reported[1] the use of a slow-gradient, sample-displacement chromatography technique pioneered by Hodges et al.[2,3] for the successful purification of a wide variety of peptides.  The key advantage of this approach is that it allows the purification of large quantities (several hundred milligrams) of peptide in a single step.

Displacement chromatography is quite an old technique.  First proposed by Tiselius in 1943 and later developed further by Horváth,[4] it involves loading the sample onto a column, and then displacing it by a constant flow of a ‘displacer’ solution which contains a compound with higher affinity for the stationary phase than any of the components.  As the displacer travels down the column, it pushes the other components downstream giving consecutive areas of highly concentrated pure substances (see Figure 1c).  While this technique allows substantially higher sample loading, the requirement for identification of a suitable displacer and the need for its subsequent removal from the final product provide substantial drawbacks to this method.

Sample displacement chromatography removes these obstacles by using the sample components themselves as displacers. During loading of the sample mixture, which is done under overload conditions, the components compete for adsorption sites on the stationary phase.  The main separation occurs during the column loading phase: the components with higher affinity will compete for sites more successfully than those with lower affinity, which will be displaced further down the column.

Rather than requiring optimization of conditions for every different peptide or molecule, a generic slow gradient of 0.1% organic modifier per minute is used to then elute the components. This approach allows excellent separation and recoveries in a single chromatographic step, and even samples of very low purity, as shown in Figure 2a, can be 'rescued' by this technique.  As described by Harris et al., all 800 mgs of the crude peptide (Figure 2a) were loaded onto a semi-preparative, reversed-phased column and subjected to slow-gradient, sample-displacement purification (Figure 2b).  Remarkably, 70 mgs of pure (>99%) peptide (Figure 2c) were obtained in a single chromatographic step.  The detailed analysis of all the fractions, presented in Figure 2d, shows the impressive separation achieved by this method.

Figure 2. a) Analytical RP-HPLC of the crude peptide. The (*) refers to the desired product. b) Semi-preparative RP-HPLC of the crude peptide under slow-gradient, sample displacement conditions. c) Analytical RP-HPLC of the purified peptide. d) Comparison of all material eluted from the semi-preparative purification.  

A wide range of synthetic peptides, including those with non-natural modifications such as biotin and carboxyfluorescein, have been successfully purified in the Brimble lab using this one-step, universal method.  Harris et al. hope that "given the data presented here, this efficient mode of HPLC purification will be embraced by the wider peptide community."

[1]  Harris, P. W. R.; Lee, D. J.; Brimble, M. A., A slow gradient approach for the purification of synthetic polypeptides by reversed phase high performance liquid chromatography. J. Pept. Sci. 2012, 18(9), 545-555.

[2]  Hodges, R. S.; Burke, T. W. L.; Mant, C. T., Preparative purification of peptides by reversed-phase chromatography: Sample displacement mode versus gradient elution mode. J. Chromatogr. 1988, 444, 349-362.

[3]  Chen, Y. X.; Mant, C. T.; Hodges, R. S., Preparative reversed-phase high-performance liquid chromatography collection efficiency for an antimicrobial peptide on columns of varying diameters (1 mm to 9.4 mm I.D.). J. Chromatogr., A 2007, 1140 (1-2), 112-120.

[4]  Horváth, C.; Nahum, A.; Frenz, J. H., High-performance displacement chromatography. J. Chromatogr., A 1981, 218, 365-393.