Ascentis Express RP-Amide HPLC columns are the most recent product additions to the Supelco HPLC product line. Combining an embedded polar group (EPG) stationary phase with the Fused-Core particles, Ascentis Express RP-Amide provides a host of useful benefits to the HPLC chromatographer. The benefits of the phase technology can be summarized as:
While the Ascentis Express C8 and C18 provide classic reversed-phase selectivity, the RP-Amide phase offers an alternative selectivity. Supelco first commercially introduced the EPG phase in 1988. At that time, large tailing factors for basic analytes continued to plague conventional C18 and C8 bonded phases. The EPG phase was found to improve peak shape of basic analytes. The early generation EPG phases were based on a two-step bonding process (Figure 1). The first step was the bonding of an aminopropylsilane to the bare silica surface creating a surface with amine functionality. In step two, palmitoyl chloride was reacted with the amine to create a long chain amide. Not all amines would be converted in the process, leaving a mixed system. These early generation EPG phases suffer from poor reproducibility.
Next generation phases, including Ascentis Express RP-Amide, are produced using a one-step process (Figure 2). In the single step process, no free amino ligands occur since the amide is introduced as a whole unit. This one-step bonding process yields excellent batch-to-batch reproducibility. Interestingly, not all EPG phases on the market use the modern, one-step bonding approach.
Figure 1. Early Generation Two-step Bonding Process for EPG Amide Phases
Figure 2. Next Generation One-step Bonding Process for EPG Amide Phases
As previously mentioned, the Ascentis Express RP-Amide phase reduces silanol interactions with basic analytes, improving peak shape. A good test to demonstrate this effect is highly basic compounds using a mobile phase pH of 7. At this pH, many of the residual silanols are in the ionized form and the basic compounds are protonated.
The protonated (charged) bases interact with the charged silanols via ion exchange and result in a tailing peak. A test mix of tricyclic antidepressants was analyzed on Ascentis Express RP-Amide and a C18 column with a mobile phase pH of 7 (Figure 3). As shown in Figure 3, the RP-Amide produces more symmetrical peaks than the C18 for these difficult test probes. Asymmetry data is summarized in Figure 4 for doxepin, imipramine, and amitriptyline.
Figure 3. Separation of Tricyclic Antidepressants on Ascentis Express RP-Amide and Conventional C18
Figure 4. Asymmetry Factors for Difficult Bases on Ascentis Express RP-Amide and Conventional C18
Ascentis Express RP-Amide provides increased selectivity for polar compounds, especially those that can act as a hydrogen bond donor. 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 5. The phenolic nature of catechols and resorcinols provides a good test for demonstrating enhanced selectivity of the RP-Amide phase. The RP-Amide phase shows complete baseline resolution of these related compounds while the C18 phase shows reduced retention, resolution, and selectivity for the phenolics. In comparing the Ascentis Express RP-Amide to the Waters™ BEH Shield RP18, a competitive EPG phase, the selectivity is very similar. The difference in this example is the Ascentis Express RP-Amide yields a backpressure half of the 1.7 μm column. This difference in backpressure means the Ascentis Express column is suitable for traditional HPLC systems while the 1.7 μm column is not.
The selectivity differences between the RP-Amide and the C18 can be a useful tool in method development. In many cases, when peaks co-elute on a C18 phase, the RP-Amide can be substituted to achieve separation without a change in mobile phase.
Figure 5.Separation of Phenolics on Ascentis Express RP-Amide & C18, & Waters BEH Shield RP18, 1.7 μm
Ascentis Express RP-Amide provides stable and reproducible analyte retention in 100% aqueous mobile phases. Many C18 phases are known to suffer from phase collapse under highly aqueous mobile phase conditions causing loss of retention. Shown in Figure 6 is a mix of organic acids analyzed under 100% aqueous conditions. Excellent selectivity and peak shape is noted for all the test probes, even citric acid, which is a notoriously difficult analyte.
Figure 6.Separation of Small Organic Acids Under 100% Aqueous Conditions
Ascentis Express RP-Amide is a blend of modern phase technology and innovative particle technology. The Fused-Core particle provides benefits in terms of speed, resolution, sensitivity, and ruggedness. The one-step RP-Amide bonding chemistry provides benefits in terms of selectivity, aqueous stability, and improved peak shape for bases.