Ascentis® Express RP-Amide Expands the Selectivity of Fused-Core ™ Particle Technology HPLC Columns

September 1, 2008
Wayne K. Way

Sigma-Aldrich/Supelco

,
William Campbell

The Application Notebook

The Application Notebook, The Application Notebook-09-01-2008, Volume 0, Issue 0

The Fused-Core particle consists of a 1.7 micron solid core and a 0.5 micron porous shell yielding a 2.7 micron diameter. One of the benefits of the Fused-Core particle is the small diffusion path (0.5 microns) compared to conventional fully porous particles. The shorter diffusion path minimizes peak broadening. In fact, there have been many reports on the vast improvements in efficiency provided by Fused-Core particles versus conventional particles. These improvements provide sub-2 micron like performance at half of the backpressure allowing Ascentis Express columns to be used in conventional HPLC as well as UHPLC systems.

Wayne K. Way and William Campbell, Supelco/Sigma-Aldrich

The Fused-Core particle consists of a 1.7 micron solid core and a 0.5 micron porous shell yielding a 2.7 micron diameter. One of the benefits of the Fused-Core particle is the small diffusion path (0.5 microns) compared to conventional fully porous particles. The shorter diffusion path minimizes peak broadening. In fact, there have been many reports on the vast improvements in efficiency provided by Fused-Core particles versus conventional particles. These improvements provide sub-2 micron like performance at half of the backpressure allowing Ascentis Express columns to be used in conventional HPLC as well as UHPLC systems.

Figure 1

While the Ascentis Express C18 provides classic reversed-phase selectivity, the Ascentis Express RP-Amide provides increased selectivity for polar compounds, especially those that can act as a hydrogen-bond donor. Phenols, carboxylic acids, and amines are just a few of the classes of analytes that have been observed having enhanced retention and selectivity on the RP-Amide phase. Other attributes of the RP-Amide include improved peak shape for bases, 100% aqueous compatibility, and low bleed for LC–MS applications.

Analyte selectivity is a function of the mechanism of interaction between the analyte and the stationary phase. With alkyl phases, the analyte retention is due primarily to Van Der Waals interactions. The Ascentis Express RP-Amide introduces a polar functionality near the silica surface, an amide.

The amide group provides for hydrogen bonding with analytes that have hydrogen bonded to a heteroatom. Phenols, carboxylic acids, amines, and, to a lesser extent, alcohols show enhanced retention on the RP-Amide phase when compared to neutral non-polar analytes. An example of the power of the hydrogen bonding mechanism is shown in Figure 1. The Ascentis Express C18 and RP-Amide columns are compared. The analyte mixture contains neutral, non-polar analytes (benzene and toluene) and protic analytes (p-methoxyphenol, p-nitrobenzoic acid, and p-chlorophenol). As observed from the chromatograms in Figure 1, the neutral molecules show slightly reduced retention on the RP-Amide, but the protic molecules show greatly enhanced retention yielding a chromatogram with very different selectivities and even a change in elution order. The potential for solving separation difficulties is tremendous.

Two other points should be noted. The Ascentis Express RP-Amide has the same high efficiency as the Ascentis Express C18 with the same low back-pressure. Secondly, both separations were carried out in the same mobile phase. This is important since it simplifies method development. If a separation is not adequate on an Ascentis Express C18, there is no need to change mobile phase to optimize the separation, simply switch to the Ascentis Express RP-Amide and if protic moieties are present, a change in selectivity will be achieved.

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