Serial versus parallel columns using isocratic elution: a comparison of multi-column approaches in mono-dimensional...

When a new separation problem is faced with high-performance liquid chromatography (HPLC), the analysis is addressed conventionally with a single column, trying to find out a single experimental condition aimed to resolve all compounds. However, in practice, the system selectivity may be insufficient to achieve full resolution. When a separation fails, the usual practice consists of introducing drastic changes in the chromatographic system (e.g. use of another column, solvent or pH). An alternative solution is taking benefit of the combined separation capability of two or more columns, which can be attained in multiple ways, such as diverse modalities of two-dimensional HPLC, or mono-dimensional HPLC with serial or parallel columns. In this work, the separation performance offered by the serial coupling of columns of different nature and length, operated at varying mobile phase composition in isocratic elution, is compared with the results offered by parallel columns. The resolution capability of both approaches is characterised through the limiting peak purities. It is demonstrated that serial columns of different lengths perform as new columns that increase enormously the probabilities of success. The potential of the approach is illustrated through the separation of 15 sulphonamides. In spite of the poor individual performance of the four selected columns (phenyl, cyano and two C18 columns, with nearly null resolution for the cyano column), it was found that the serial coupling of the phenyl and cyano columns of appropriate lengths succeeded in the full resolution of the 15 compounds in 20-25min, and the serial coupling of the two C18 columns yielded acceptable resolution in less than 20min.