Improved Resolution and Retention of Highly Acidic and Highly Basic Molecules Using Scherzo SM-C18 Multi-Mode Column

The Application Notebook

The Application Notebook, The Application Notebook-02-01-2013, Volume 0, Issue 0

Imtakt USA

Bhavana Verma and Itaru Yazawa, Imtakt USA

Highly charged basic and acidic compounds are analyzed in many different fields and industries, including the fields of biochemistry, food chemistry, and pharmaceuticals. It is difficult to retain and resolve these types of compounds on a standard C18 HPLC column. In order to overcome these separation challenges, Imtakt Corporation has introduced the Scherzo SM-C18 column, which has a novel multi-mode stationary phase containing anion, cation, and C18 ligands. The very acidic molecule inositol hexaphosphate (phytic acid) and the very basic molecules spermine and spermidine are retained much better by the Scherzo SM-C18 column than by a standard C18. In addition, although many multi-mode HPLC columns suffer from weak batch-to-batch reproducibility, the Scherzo SM-C18 column shows excellent reproducibility for a range of pharmaceutical compounds.

Experimental

All data was generated with a semi-micro HPLC system equipped with ELSD detection. Inositol hexaphosphate (phytic acid) was eluted on a Scherzo SM-C18 column of dimensions 50 mm × 3 mm (see Figure 1). Gradient elution was utilized with a flow rate of 0.4 mL/min, column temperature of 37 °C, and pressure of 50–60 bar.

Figure 1: Elution of inositol hexaphosphate (phytic acid).

Spermine and spermidine were eluted on a Scherzo SM-C18 column of dimensions 75 mm × 3 mm (see Figure 2). Gradient elution was utilized with a flow rate of 0.4 mL/min, column temperature of 37 °C, and pressure of 40 bar.

Figure 2: Elution of spermine and spermidine.

Results and Discussion

The Scherzo SM-C18 multi-mode HPLC column easily retains highly charged basic/acid compounds that are not retained on a standard C18 column. This column can also recognize slight structural differences: the very similar spermine and spermidine were well resolved on the Scherzo SM-C18 column, while they coeluted on a standard C18 column. Not only does the Scherzo SM-C18 exhibit excellent resolution and retention, it also shows great batch-to-batch reproducibility for a wide range of small molecules, offering significant advantages for method development.

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