Mixed-mode chromatography (MMC) is a type of chromatographic method in which multiple-mode take place between the stationary phase and solute in the feed. MMC has been found to have many advantages and thus became a research hot point. However, before going wide applications, the synthesis of new MMC column and/or the selection of some commercial columns having the character of MMC need to be established. Based on the fact that some commercial ion-exchange columns perform the separation behavior of hydrophobic interaction chromatography (HIC), one of four commercial weak-cation exchange (WCX) column, a two-dimensional mode (WCX, HIC) column called as 2D column, and standard proteins were selected to investigate and compare their retention behaviors. The two type columns have some common characters: proteins can be separated by either WCX, or HIC mode, but have some quite different column efficiencies, and the profiles of “U-shape” elution curve. The width of the “U-shape” curve was experimentally found to relate to the chromatographically dynamic factor, i.e., the broader the “U-shape” curve, the narrower the chromatographic peak obtains, while the magnitude of the critical point of the “U-shape” curve relates to the protein retention, a larger value of a critical point corresponds to a stronger retention. An intensive comparison between the two types of column efficiency was carried out by the peak capacity of proteins. The experimental results showed that the 2D column has much more number of peak capacity than the commercial WCX column, either for one-dimensional, or two-dimensional separation. However, for the separation of a group proteins, the elution order is dominated by the comprehensive consideration of the peak capacity, the dynamic, and thermodynamic factors. Finally the nomenclature of the various stationary phases of MMC and/or 2D column employed in two-dimensional liquid chromatography were suggested. Although, in terms of column efficiency, none of the four commercial WCX column could be compared to the 2D column, at least, two of them as “quasi-2D column” may be employed for protein separation with both of WCX and HIC modes by means of either off-line in all cases, or on-line in some cases. However, none of them has been found to approach to the criteria of the 2D column. The nomenclature and expressions of MMC and 2D columns were suggested and the difference between the MMC and 2D columns were also explained.

Key words: two-dimensional chromatographic stationary phase, mixed-mode chromatography, protein separation, ion-exchange chromatography, hydrophobic interaction chromatography, stoichiometric displacement theory