Mixed-mode chromatography

Mixed-mode chromatography (MMC), or multimodal chromatography, refers to chromatographic methods that utilize more than one form of interaction between the stationary phase and analytes in order to achieve their separation. What is distinct from conventional single-mode chromatography is that the secondary interactions in MMC cannot be too weak, and thus they also contribute to the retention of the solutes. Mixed-mode chromatography (MMC), or multimodal chromatography, refers to chromatographic methods that utilize more than one form of interaction between the stationary phase and analytes in order to achieve their separation. What is distinct from conventional single-mode chromatography is that the secondary interactions in MMC cannot be too weak, and thus they also contribute to the retention of the solutes. Before MMC was considered as a chromatographic approach, secondary interactions were generally believed to be the main cause of peak tailing. However, it was discovered afterwards that secondary interactions can be applied for improving separation power. In 1986, Regnier’s group firstly synthesized a stationary phase that had characteristics of anion exchange chromatography (AEX) and hydrophobic interaction chromatography (HIC) on protein separation. In 1998, a new form of MMC, hydrophobic charge induction chromatography (HCIC), was proposed by Burton and Harding. In the same year, conjoint liquid chromatography (CLC), which combines different types of monolithic convective interaction media (CIM) disks in the same housing, was introduced by Štrancar et al. In 1999, Yates’ group loaded strong-cation exchange (SCX) and reversed phase liquid chromatography (RPLC) stationary phases sequentially into a capillary column coupled with tandem mass spectrometry (MS/MS) in the analysis of peptides, which became one of the most efficient technique in proteomics afterwards. In 2009, Geng’s group first achieved online two-dimensional (2D) separation of intact proteins using a single column possessing separation features of weak-cation exchange chromatography (WCX) and HIC (termed as two-dimensional liquid chromatography using a single column, (2D-LC-1C). Higher selectivity: for example, positive, negative and neutral substances could be separated by a reversed phase (RP)/anion-cation exchange (ACE) column in a single run. Higher loading capacity,for example, loading capacity of ACE/ hydrophilic interaction chromatography (HILIC) increased 10-100 times when compared with RPLC,which offered a new selection and idea for developing semi-preparative and preparative chromatography.