The influence of injection volume on efficiency of microbore liquid chromatography columns for gradient and isocratic...

The injection volume and the associated column volume overload is one of the most common issues in miniaturized chromatography. The injection volume should not exceed 10% of the effective column volume. A further reduction of the injection volume leads to an increase in chromatographic efficiency. However, the signal intensity must be above a certain threshold to generate a chromatographic peak that can be detected. Therefore, the injection volume has to be optimized to reach the ideal balance between chromatographic efficiency and sensitivity. This study examined the general influence of the injection volume for both isocratic and gradient elution, depending on the retention factor and peak standard deviation. For this purpose, substances of different polarity were selected to represent a broad elution spectrum. Besides the model analyte naphthalene, these were mainly pharmaceuticals. For all measurements a microbore column with an ID of 300 µm and packed with 1.9 μm fully porous particles was used. For isocratic elution, the injection volume was varied between 4 and 16% of the effective column volume. The retention factors were adjusted between 2 and 10. For gradient elution, the injection volume was varied between 4 and 160% of the effective column volume. The observed effects were further investigated using the gradient kinetic plot theory. In isocratic elution, a loss in plate height up to 50% was observed for components that elute near the void time. A significant reduction of the chromatographic efficiency was noticed up to a retention factor of 4. In gradient elution, a reduction in peak capacity could only be observed if the injection volume exceeded 40% of the effective column volume. For some substances, only a slight loss in peak capacity was noticed even with a volume overload of 160%.