27
Figure 11. Separation of 12 amino acids
Figure 13. 2D Property of the complex mixture in 2D plane and its separation by reverse-phase, ion-exchange, and mixed- mode chromatography mode
Figure 12. Separation of monocarboxylic acids
Case Studies for Mixed-Mode Chromatography
1. Analysis of Amines and Amino Acids in Reversed-Phase Ion-Exchange Modes Amines and amino acids are used as building blocks in many chemical and pharmaceutical productions. These molecules are hydrophilic in nature. Several methods are used to analyse amines and amino acids. In one approach, derivatisation is used to convert hydrophilic molecules with low UV activity to more hydrophobic molecules with UV activity [7]. Ion-pairing reagent, in combination with regular reversed-phase chromatography, provides good retention and selectivity, and is proven to be a robust approach [8]. Unfortunately, ion-pairing chromatography is not compatible with LC/MS and preparative chromatography. Mixed-mode stationary phase can be considered as ion-pairing reagent attached to the surface. In mixed- mode columns, significant retention occurs via the cation-exchange sites of the stationary phase. At lower pH, amines are always positively charged and amino acids are basic in nature. Additionally, at lower pH, carboxylic acid functionality of amino acids is not ionised and amino acids will retain by the combination of reversed-phase and cation- exchange mechanism (Figure 11). The only requirement is that acidic functionality of stationary phase remains ionised at lower pH
(pKa below 3). Mixed-mode columns with strong acidic functionalities on the surface allow a wide range of mobile phase composition and pH providing enhanced cation-exchange interactions. If pH of the mobile phase is lower than pKa of the acidic group on the column surface, the ion- exchange mechanism diminishes, which makes the mixed-mode phase similar to reversed-phase when tested with all type of compounds.
2. Analysis of Organic and Inorganic Acids in Reversed-phase Ion-Exchange Modes Two approaches exist in analysis of acidic compounds. The first includes retention by reversed-phase and anion-exchange mechanism - pH of the mobile phase in this case needs to be above the pKa of the acids (Figure 12). This will ionise the analyte and will increase anion-exchange interaction between analyte and stationary phase. In the second approach, mobile phase pH is lower than pKa of the acid. The acid becomes non- ionised and more hydrophobic, and it is retained by reversed-phase mechanism [9]
3. Analysis of Complex Mixtures in Mixed-Mode Complex mixtures are common in pharmaceutical formulations. Such mixtures contain acidic, basic organic, and inorganic compounds, and hydrophobic and polar compounds. Presence of multiple compounds and related impurities requires
additional selectivity of separation. Mixed- mode stationary phase offers a 2D selectivity on a single column (Figure 13). Small difference in hydrophobic or/and ionic properties usually provide sufficient selectivity when analysed on mixed-mode column. Adjustment of organic modifier or buffer concentration allows for enhancement or suppression of hydrophobic or ionic interaction. The mobile phase pH selection is another powerful tool for selectivity control (Figure 14), providing strong effect on relative retention of charged species.
Figure 14. Separation of complex mixture on Primesep 200
4. Analysis of Polar Neutral and Ionic Compounds in HILIC/Mixed-Mode HILIC on bare-silica and aminopropyl columns can be considered as early cases of weak cation-exchange/HILIC and weak anion-exchange/HILIC [10]. In order to see cation-exchange effect of silanols, the pH of
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