A Weak Anion-Exchange/Reversed-Phase Mixed-Mode HPLC Column and its Applications

Mixed-mode columns address key challenges in analytical method development for HPLC. Reversed-phase (RP) columns (e.g., C18) are most widely used for HPLC separations.1 However, their use is limited by dewetting in highly aqueous environments,2,3 peak tailing for basic molecules4–6 and insufficient retention of hydrophilic compounds (e.g., organic acids). Ion exchange (IEX) chromatography is used to separate ionic or ionizable molecules.7 However, IEX is of limited utility in separating small-molecules differing only in hydrophobicity. Ion pairing chromatography can help to overcome the aforementioned difficulties, but it often requires extended equilibration time, a complicated mobile phase that is incompatible with MS, and a dedicated column.8 Mixed-mode chromatography combines aspects of IEX and RP chromatography. The combination of both hydrophobic and ion-exchange properties facilitates the independent control of retention for ionizable and neutral molecules. Many applications involving hydrophilic ionizable compounds that are problematic on a C18 column are easily addressed on a mixed-mode column.

Figure 1 - Surface chemistries of mixed-mode stationary phases.

Mixed-mode stationary phases can be classified into four categories based on column chemistry (Figure 1). The Type I material includes a blend of two different stationary phases (RP and IEX). Type II packing involves bonded silica modified by a mixture of both RP and IEX ligands in the bonding step. Although these two materials seem straightforward, their use in many applications is limited by selectivity drifting, mainly due to the difference in hydrolytic stability of RP and IEX ligand linkages to the silica substrate. The new mixed-mode stationary phases address this using functional silyl ligands that bear both RP and IEX functionalities (Types III and IV). While the constant ratio between RP and IEX bonded sites greatly improves the selectivity ruggedness in both materials, a pronounced distinction exists: Type III material is silica functionalized with IEX-embedded alkyl silyl ligands, and can be viewed as an IEX-modified RP packing.9 In comparison, Type IV material involves bonding IEX-tipped alkyl silane ligand to the silica substrate. This emphasizes the IEX property, making this in essence an RP-modified IEX packing.10

This article describes a weak anion-exchange (WAX)/RP mixed-mode silica column, the Acclaim® Mixed-Mode WAX-1 column (Dionex Corp., Sunnyvale, CA), including its column chemistry, chromatographic properties, and example applications.

Column features

Figure 2 - Acclaim Mixed-Mode WAX-1 column.

The columns are packed with a silica-based stationary phase that incorporates both hydrophobic and weak anion-exchange properties (Figure 2). Unlike conventional RP material, this packing features a hydrophobic alkyl chain with a tertiary amine terminus (Type IV mixed-mode material), providing superior flexibility for method development.

Adjustable selectivity

Figure 3 - Adjustable selectivity, ionic strength. Column: 4.6 × 150 mm Acclaim Mixed-Mode WAX-1 column, 5 μm (same below unless stated otherwise); 50/50 v/v acetonitrile/phosphate buffer; temperature: 30 °C; flow rate: 1 mL/min; injection volume: 2 μL; detection: UV at 210 nm. Peaks: 1) butylbenzene (0.1 mg/mL), 2) 4-hydroxybenzoic acid (0.5 mg/mL).

Figure 4 - Adjustable selectivity, pH: Conditions as in Figure 3 using 50/50 v/v acetonitrile/20 mM phosphate in the mobile phase.

Figure 5 - Adjustable selectivity, organic content: Conditions as in Figure 3 using acetonitrile/phosphate at pH 6.

With adjustable selectivity, simple changes in mobile phase composition can ensure optimum separations. The Acclaim Mixed-Mode WAX-1 column combines both hydrophobic and anion-exchange characteristics, which facilitates easy selectivity adjustments through changes in mobile phase ionic strength, pH, or organic content, either independently or concurrently (Figures 3–5). Increased ionic strength results in decreased, increased, and unchanged retention for acidic, basic, and neutral molecules, respectively. Hydrophobic retention is markedly affected by organic content. In general, all molecule types (acids, bases, and neutrals) are less retained on this column with increased organic content in the mobile phase, when keeping other conditions constant (e.g., ionic strength, pH, temperature, etc.). Although pH has little effect on the retention of neutral molecules, it significantly affects retention of some anions. For example, lowering pH decreases negative charge in carboxylate-containing molecules, giving rise to decreased ion-exchange retention.

Selectivity orthogonal to reversed-phase columns

Figure 6 - Orthogonal selectivity of the Acclaim Mixed-Mode WAX-1 column versus a C18 RP column; 50/50 v/v acetonitrile/50 mM phosphate buffer, pH 2.8, for the Mixed-Mode WAX-1 column and 20/80 v/v acetonitrile/50 mM phosphate buffer, pH 2.8, for the Acclaim 120 C18 column; UV detection at 220 nm. Peaks (0.2 mg/mL for all): 1) benzoic acid, 2) phthalic acid, 3) 1,2,3-tricarboxylicbenzene.

During drug development, a secondary LC method with dramatically different or orthogonal selectivity is often required to complement the primary method, which typically uses an RP column. As shown in Figure 6, the column chemistry of the Acclaim Mixed-Mode WAX-1 column meets this requirement by providing different elution order for organic acids compared to a conventional RP column.

Ideal selectivity for anionic molecules

Figure 7 - Separation of mono-carboxylic acids. Conditions: mobile phase, 25 mM phosphate buffer, pH 6.0; temperature, 30 °C; flow rate, 0.8 mL/min; injection volume, 10 μL; detection, UV at 210 nm. Peaks: 1) quinic acid, 2) shikimic acid, 3) glycolic acid, 4) lactic acid, 5) acetic acid, 6) formic acid, 7) ascorbic acid, 8) isoascorbic acid, 9) propionic acid.

Although organic acids can sometimes be separated on an RP column in ion-suppression mode at low pH, dewetting often causes sudden, reversible retention loss in highly aqueous conditions. Even aqueous-compatible RP columns fail to separate hydrophilic organic acids, primarily because hydrophobic retention alone is inadequate to differentiate molecules with similar hydrophobicities. The Acclaim Mixed-Mode WAX-1 column features a modified anion-exchange stationary phase (Type IV) that provides not only adequate retention, but also ideal selectivity for a variety of anionic molecules. It is effective even for weakly charged anions such as hydrophilic monocarboxylic acids, which would be very difficult to separate on any RP column (Figure 7). It outperforms commercial Type III mixed-mode columns for the separation of organic acids: Just as basic analytes exhibit peak tailing on a silica-based RP column, due to the secondary interaction between positively charged analytes and the negatively charged, unreacted silanols, acidic analytes tail on a Type III mixed-mode column due to the secondary interaction between negatively charged analytes and the positively charged embedded anion-exchange functional groups.