Feed injection in liquid chromatography: Reducing the effect of large-volume injections from purely organic diluents in reversed-phase liquid chromatography

In liquid chromatography (LC), discrepancies in liquid properties such as elution strength and viscosity lead to a mismatch between the sample diluent and mobile phase. This mismatch can result in peak deformation, including peak splitting or even breakthrough, particularly when large sample volumes are injected. The formation of a T-junction between sample solution and mobile phase flow stream, a technique previously used in supercritical fluid chromatography, is the key enabler of feed injection in LC. This T-junction allows the injection needle to infuse the sample directly into the mobile phase. It ensures that the diluent is continuously mixed with the mobile phase before introduced onto the column, thereby reducing the initial solvent mismatch. The degree of dilution depends on the ratio between mobile phase flow rate (Q_mp) and feed rate (Q_feed) at which the sample is infused. Our study examined the effect of several parameters on the feed injection of large sample volumes from purely organic diluents in reversed-phase LC. These parameters included the type of diluent, compound retention factor (k), injected sample volume (V_inj), and Q_mp. With varied Q_feed, all compounds revealed a similar range of optimal values for Q_r = (Q_mp-Q_feed)/Q_feed between 2 and 5, a range unaffected by V_inj and Q_mp. For Q_r > 5, the slope of the plate height curves (H vs. Q_r) decreases with increasing k, potentially extending the range of optimal Q_r-values. However, the best Q_r-value for a separation is determined by the compound with the smallest k, simplifying optimization. Using feed injection, we were able to reduce plate heights by up to a factor of 8 compared to classic flow-through injection of large sample volumes.