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Figure 2. Ion-exchange and reverse phase mechanism in separation of acids, bases, amino acids, and neutral compounds
Figure 4. Loading study for a purification of a polar amino compound on analytical column.
Figure 3. LC/MS and prep compatible separation of catecholamines
groups as a part of hydrophobic ligand attached to the surface. These columns show better reproducibility and retention control than the first generation. Several studies successfully demonstrated applicability of these columns to retention of neutral and ionic compounds [4]. These stationary phases are used for analysis of acidic, basic and neutral compounds. In case of dual interaction, ions opposite the stationary phase are retained by ion-exchange interaction [5]. Hydrophilic acidic compounds are usually transformed into a non-ionised form, reducing compound polarity. A typical example includes analysis of basic, acidic and neutral compounds in one run at lower pH, when carboxylic groups are not ionised and the acidic compound is retained by reversed-phase interaction (Figure 2). Two main approaches of third-generation (Figure 1c) mixed-mode columns are popularised by the SIELC and Dionex companies.
Dionex columns consist of ion-exchange
Figure 5. Simultaneous separation of Na+ and CI- ions
beads placed at the surface of silica gel, providing one of the ion-exchange interactions. The second ionic interaction, as well as hydrophobic interaction, comes from the hydrophobic ligand attached to the surface. SIELC columns have a ligand that carries cation- and anion-exchange properties, as well as a hydrophobic chain assembled as one ligand, which is attached to the surface of silica.
Third-generation mixed-mode columns have increased capacity and retention control. Ionic compounds can be retained by cation- and anion-exchange mechanism, and the hydrophobic compounds by reversed-phase mechanism.
The main advantage of this stationary phase configuration is an ability to retain both polar cations and anions at the same time with low buffer concentration in the MP. Ionisation state of stationary phase and ratio between cation-exchange and anion-exchange sites can be adjusted by changing the pH of the mobile phase.
Benefits of Mixed-Mode Chromatography Multiple controllable interactions on a column allow better control of retention for various analytes. Polar ionic and non-polar non-ionic compounds can be separated in a single run. Selectivity of separation can be controlled by varying the amounts of organic
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