Mixed mode chromatography combines aspects of ion exchange chromatography and conventional reversed-phase (RP) chromatography. The combination of both hydrophobic and ion-exchange properties allows for independent control of retention for ionizable and neutral molecules.
Mixed mode chromatography combines aspects of ion exchange chromatography and conventional reversed-phase (RP) chromatography. The combination of both hydrophobic and ion-exchange properties allows for independent control of retention for ionizable and neutral molecules. Therefore, many application challenges involving hydrophilic ionizable compounds that would be difficult for C18 columns, can be easily tackled on a mixed-mode column. We have developed a new silica-based mixed-mode stationary phase, Acclaim Mixed-Mode WAX, which provides both weak anion-exchange and RP characteristics facilitated by covalently attaching a hydrophobic alkyl chain with an ionizable terminus to the silica surface. The new column features complete control of selectivity, orthogonal selectivity to RP columns, high capacity and unique selectivity for anionic molecules and multi-mode retention mechanisms (i.e., RP, anion-exchange and HILIC).
Acclaim Mixed-Mode WAX (4.6 × 150 mm, dp = 5 ⛤m, Dionex, Sunnyvale, California, USA) columns were used for the separation. Separations were performed on a modular Summit HPLC system (Dionex) equipped with a P680 gradient pump, ASI-100 Autosampler, TCC-100 column oven and UVD 340 detector. Chromeleon 6.70 Chromatography Management Software (Dionex) was used for system control and data processing. All standards were purchased from Sigma-Aldrich (St Louis, Missouri, USA). The chromatographic conditions are given in Figure 1.
Figure 1 shows separation of a mixture of basic, neutral and acidic pharmaceutical-related compounds using a gradient method. We used a commonly employed mobile phase system containing acetonitrile, D.I. water and a phosphate buffer. All components in the mixture elute not only with symmetrical peaks, but also with good resolution. Unlike conventional RP columns, the unique column chemistry of the Acclaim Mixed-Mode WAX allows for fast elution of basic molecules with symmetrical peaks, high capacity and good selectivity for anionic molecules, such as acidic drugs and highly hydrophilic organic acids. Meanwhile, neutral molecules, such as steroids retained by hydrophobic interaction, usually elute after bases but before acidic molecules at pH 6. Thus, the separation is easily optimized by adjusting mobile phase ionic strength, pH and organic modifier, either independently or concurrently.
The Acclaim Mixed-Mode WAX column provides unique and adjustable selectivity, excellent peak shape for all types of molecules (i.e., acids, bases and neutrals) and multi-mode retention mechanism (i.e., RP, anion-exchange and HILIC). Therefore, it can be used as either a primary column for a broad range of applications including pharmaceuticals, food and beverage, chemical or a complementary column (to conventional RP columns) that provides orthogonal selectivity.
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