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Figure 6. Analysis of Neutral hydrophobic compounds on Scherzo SM-C18 mixed-mode column
Figure 7. Simultaneous separation of acidic, neutral, and basic pharmaceuticals
Figure 8. Effect of acetonitrile and buffer concentration on retention of basic drug and counter-ions (tri-modal column)
reagents for polar compounds that are compatible with LC/MS and prep chromatography (Figure 3). Large numbers of ion-exchange sites on the stationary phase increases loadability of the column (Figure 4). Symmetrical peak shape is often observed for basic molecules. First-generation mixed- mode column (with ion-exchange interaction coming from silanols), made it easy to overload weak silanol groups, resulting in peak tailing [6].
Figure 9. Separation of anilines on mixed-mode Primesep 200 column
Another benefit of mixed-mode chromatography is that the same column can be used either in a single mode (cation- exchange, anion-exchange, reversed-phase), or in combination of modes. Sodium and chloride ions can be separated in pure ion- exchange node on Trinity mixed-mode columns (Figure 5). Mixed-mode columns can be used in pure reversed-phase mode for separation of neutral hydrophobic compounds (Figure 6). Mixed-mode columns provide same efficiency as regular reversed- phase columns, with plate count achieving 100K/meter for 5um particles. Same columns were also used in separation of more complex mixtures, including hydrophilic ionised, hydrophobic ionised, and hydrophobic neutral compounds. This approach can be used for analysis of drugs and corresponding counter-ions, pharmaceutical formulations, and other complex mixtures (Figures 7, 8). Retention times for some compounds can be adjusted independently to facilitate different selectivity and even to control the order of elution.
Figure 10. Separation of diostreomers (quinidine/quinine) on mixed-mode column
component, buffer pH, and buffer concentration. Contrary to reverse-phase chromatography, buffer pH will affect not only ionisation state of analytes, but also the ionisation state of stationary phase. This is
because the ionic strength of the column can be increased or decreased based on the pH of the mobile phase. Additional ion- exchange interactions allow the development of methods without ion-pairing
Complex interaction of mixed-mode column with analytes often produced superior selectivity as compared to single mode column case. Structural and diostereoisomers of compounds with charged functional groups can be resolved easily with mixed-mode approach (Figures 9, 10)
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