The Separation of Closely Related Compounds Using Ascentis Express F5

February 11, 2011

The Application Notebook

The Application Notebook, The Application Notebook-02-11-2011, Volume 0, Issue 0

The separation of closely related compounds is of interest to many disciplines including natural products, flavors and fragrances, and pharmaceuticals.

David S. Bell and Carmen T. Santasania, Supelco/Sigma-Aldrich

The separation of closely related compounds is of interest to many disciplines including natural products, flavors and fragrances, and pharmaceuticals. The Ascentis® Express F5 HPLC columns have a unique mix of shape selectivity, ionic character, and hydrophobic retention to provide the selectivity needed for the separation of closely related compounds.

Ascentis Express is a high-speed, HPLC column based on Fused-Core® particle technology. The F5 bonded phase exhibits strong dipole potential (polar interaction) from the carbon-fluorine bonds, pi-pi interaction potential, and the ability to interact via charge-transfer interactions due to the electronegativity of the fluorine atoms. The rigidity of the F5 phase is believed to provide enhanced shape selectivity of analytes differing in size and spatial attributes. Chromatographers are often faced with the challenge of separating compounds that are very similar in their solubility. Separation on nonpolar phases such as C18 is driven by differential partitioning of analytes, therefore, the alkyl phases are often ineffective in meeting this challenge. Hydrocortisone and prednisolone (see Figure 1) differ by a single double bond. Their solubility is very similar; however, their shapes differ significantly. Figure 2 shows a comparison of their separation, along with prednisone internal standard (IS), using both a C18 and an F5 stationary phase. The F5 phase is shown to provide the separation of these closely related compounds.

Figure 1: Structures of Hydrocortisone and Prednisolone.


F5 phase exhibits increased ionic and polar interactions relative to common alkyl phases. The rigidity of the F5 phase also provides increased shape selectivity over commonly used alkyl phases. These alternative mechanisms of retention often provide selectivity not readily achieved on the more traditional phases. Retention and selectivity of highly polar and ionic species as well as separation of closely related neutral compounds have been used to demonstrate the power of the F5 phase. The combination of the selectivity provided by the F5 phase and the efficiency of Fused-Core particles provides even greater resolving power.

Figure 2: Comparison of C18 and F5 for the separation of closely related steroids. Column(s): Ascentis Express F5 (53590-U) and Ascentis Express C18 (53827-U), 10 cm × 4.6 mm, I.D., 2.7 µm particle size, mobile phase A: water, mobile phase B: methanol, mobile phase ratio: A:B 50:50, v/v, flow rate: 0.8 mL/min., temp.: 35 °C, det.: UV at 240 nm, Peaks: 1. Hydrocortisone, 2. Prednisolone, 3. Prednisone (IS).

Trademarks: Ascentis — Sigma-Aldrich Biotechnology LP; Fused-Core — Advanced Materials Technology, Inc.


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