A Weak Cation-Exchange, Reversed-Phase Mixed-Mode HPLC Column and its Applications

Basic compounds are widely used in many areas, such as pharmaceutical development, biological research, consumer products, and in the chemical industry. Although reversed-phase (RP) liquid chromatography is the most commonly used analytical separation technique for a broad spectrum of applications, the task becomes highly challenging when applied to basic analytes. Basic molecules often elute as tailing peaks at mid-pH (i.e., pH 7) on RP silica columns, mainly due to the secondary interaction resulting from residual silanol groups on the silica surface. This adverse effect is minimized by both increasing surface coverage and by using high-purity silica gel. However, the resulting material is incompatible with highly aqueous mobile phases because of the dewetting phenomenon on a hydrophobic surface.1

Another challenge relates to inadequate retention of hydrophilic, basic molecules on an RP column. Many pharmaceuticals are hydrophilic, basic molecules. Very often, an RP column fails to give sufficient retention because of the hydrophilicity of such compounds. Although ion-pairing chromatography increases retention for hydrophilic charged molecules, it often requires a long equilibration time, a dedicated column, and a complicated mobile phase that is incompatible with a mass spectrometer. Conventional ion-exchange (IEX) liquid chromatography is often used to separate charged molecules, but it is not suitable for separating neutral molecules because conventional IEX columns provide inadequate hydrophobic retention.

Mixed-mode chromatography is a combination of IEX and RP chromatography. A typical mixed-mode stationary phase has both hydrophobic and ion-exchange properties and facilitates the independent control of retention for ionizable and neutral molecules. Therefore, many application challenges involving hydrophilic ionizable compounds that are difficult for an RP column can be easily resolved on a mixed-mode column. Mixed-mode stationary phases can be grouped into four categories according to the column chemistry: type I—blend of two different stationary phases (RP and IEX materials), type II—silica modified with a mixture of RP and IEX ligands, type III—silica modified with ion-exchange functionality embedded alkyl ligands, and type IV—silica modified with ion-exchange functionality tipped alkyl ligands. Type I and II materials exhibit undesirable selectivity drifting due to the different hydrolytic stability between the RP ligand bonded sites and IEX ligand bonded sites. Type III and IV materials provide greatly improved selectivity ruggedness because of the constant ratio between RP and IEX bonded sites. The main difference between type III and IV materials is that the former highlights the RP property and can be viewed as an IEX-modified RP packing, while the latter emphasizes the IEX property and features an RP-modified IEX packing. As a result, type IV mixed-mode materials offer several advantages for separating mixtures containing ionic and ionizable molecules (see below).

Figure 1 - Acclaim mixed-mode columns.

Acclaim® mixed-mode columns (Dionex Corp., Sunnyvale, CA) are based on type IV chemistry, and currently contain three types: the Acclaim Mixed-Mode WAX-1,2 Acclaim Mixed-Mode HILIC-1,3 and Acclaim Mixed-Mode WCX-1.4 Their column chemistries are illustrated in Figure 1. All three columns feature a long alkyl chain with polar terminus–tertiary amine, diol, and carboxylate functionalities for Mixed-Mode WAX- 1, Mixed-Mode HILIC-1, and Mixed-Mode WCX-1, respectively. This article focuses on the Acclaim Mixed-Mode WCX-1 column, including column chemistry, chromatographic properties, and applications. 

Column overview

Acclaim Mixed-Mode WCX-1 columns are packed with a silica-based stationary phase that incorporates both hydrophobic and weak cation-exchange (WCX) properties (Figure 1c). Unlike conventional RP material, the packing has a hydrophobic alkyl chain with a carboxylate terminus and offers the potential for a wide range of applications. Its main features include adjustable selectivity; orthogonal selectivity; selectivity for basic molecules; and multimode retention mechanisms—reversed-phase, cation-exchange, anion-exclusion, and hydrophilic interaction chromatography (HILIC).

Adjustable selectivity

Figure 2 - Adjustable selectivity—ionic strength effect.

Figure 3 - Adjustable selectivity—pH effect.

Selectivity is the most important factor in a separation. Although RP columns (i.e., C18) are most commonly used for small molecule separation, their selectivities are rather similar. The Acclaim Mixed-Mode WCX-1 combines both hydrophobic and cation-exchange characteristics so that its selectivity can be manipulated in order for the retention magnitude of each retention mode to be adjusted by changing mobile phase ionic strength, pH, and the organic solvent content, either individually or concurrently. With increasing ionic strength (cation concentration), the retention decreases for cationic analytes with virtually no change for neutral analytes (Figure 2). Because of its weak cation-exchange property, cation-exchange interaction can be switched on or off by changing the pH (see Figure 3). At pH 6.5, the carboxyl group on the stationary phase is negatively charged; thus both cation-exchange and hydrophobic interaction contribute to the retention. As a result, benzoic acid elutes first due to electrorepulsion, and benzyl amine elutes later because of the combination of electroattraction and hydrophobic retention. At pH 2.8, on the other hand, the carboxyl group on the stationary phase is protonated and exhibits no cation-exchange capacity. As a result, protonated benzyl amine elutes first due to its low hydrophobicity, uncharged benzoic acid becomes more retained, and there is virtually no change for the neutral molecule (naphthalene). Organic solvent content in the mobile phase affects hydrophobic interaction; thus selectivity change can be achieved accordingly.