In-Loop Analyte Degradation in Two-Dimensional Liquid Chromatography: Example and Solutions

The use of two-dimensional liquid chromatography (2D-LC) to assess peak purity is becoming common in the pharmaceutical industry. In this application space, it is critically valuable to demonstrate that all compounds eluted from a column during an LC assay are accounted for, and that no impurities are “hiding” under the peaks of known compounds. Although the addition of the second dimension (2D) separation makes 2D-LC an exquisitely powerful tool, it also warrants additional care when interpreting the results to avoid misidentification of apparently new peaks as coeluted impurities, when in fact they are analytical artifacts. In this installment of “LC Troubleshooting,” we describe one such possible artifact that can arise because of the degradation of compounds during the transfer of fractions of the first dimension (1D) column effluent to the 2D separation. We suggest simple experiments to determine whether new peaks observed in 2D chromatograms result from degradation, ultimately increasing our confidence in the interpretation of 2D-LC results.