Apparent retention volume variation with flow rate change in high performance liquid chromatography

Recent studies have demonstrated noticeable flow rate dependency of the chromatographic zone retention volume with respect to migration within the empty capillary. This appears to be the result of superposition of asymmetric lateral diffusion and of the laminar flow profile. Although these effects have been studied on empty capillaries, in the presence of the packed column, the retention shift may be insignificant relative to the adsorption-based retention of the analytes. In the case of fast and ultrafast HPLC with short capillary columns, the effect of extra-column caused variation in the analyte retention may constitute an increase of up to 120 % of the overall retardation. Small columns have very small column void volume, e.g. 1.0 x 50 mm with a column void volume of 24 μl, where the extra column volume within the connecting capillary can be as great as 185 μl. This great difference in volume, especially considering that some systems contain even longer connecting tubing for 2 dimensional HPLC or LC-MS systems, can demonstrate a significant shift in the overall retardation and may cause identification and quantitation problems. Experiments were done with common mobile phase solvents and readily available peek tubing at different variation of length and inner diameter. The origin of the phenomena is discussed, as well as the main influencing parameters such as capillary material, internal diameter, type and composition of the mobile phase. This research illustrates the importance of extra column volume on the overall separation in HPLC. The degree of band broadening and the apparent increase in retention volume is driven by the laminar flow profile and concomitant diffusion between the layers within the connective tubing of the HPLC system. The process of molecular diffusion alone has been shown to have negligible impact on this effect and is a positive outcome for systems requiring “parking” within sample loops such as in LC x LC systems. However, the deformation of sample plugs due to laminar flow effects were greatest at higher flow rates and in narrower tubing, which could have a significant impact on fast LC technologies such as UHPLC, short and narrow columns, and systems with unavoidable additional tubing lengths. This effect should be considered during method development and transfers between HPLC systems with variable extra column tubing dimensions and especially when utilizing micro columns with non-porous particles or in cases of minimally retentive analytes

Apparent retention volume variation with flow rate change in high performance liquid chromatography

Recent studies have demonstrated noticeable flow rate dependency of the chromatographic zone retention volume with respect to migration within the empty capillary. This appears to be the result of superposition of asymmetric lateral diffusion and of the laminar flow profile. Although these effects have been studied on empty capillaries, in the presence of the packed column, the retention shift may be insignificant relative to the adsorption-based retention of the analytes. In the case of fast and ultrafast HPLC with short capillary columns, the effect of extra-column caused variation in the analyte retention may constitute an increase of up to 120 % of the overall retardation. Small columns have very small column void volume, e.g. 1.0 x 50 mm with a column void volume of 24 μl, where the extra column volume within the connecting capillary can be as great as 185 μl. This great difference in volume, especially considering that some systems contain even longer connecting tubing for 2 dimensional HPLC or LC-MS systems, can demonstrate a significant shift in the overall retardation and may cause identification and quantitation problems. Experiments were done with common mobile phase solvents and readily available peek tubing at different variation of length and inner diameter. The origin of the phenomena is discussed, as well as the main influencing parameters such as capillary material, internal diameter, type and composition of the mobile phase. This research illustrates the importance of extra column volume on the overall separation in HPLC. The degree of band broadening and the apparent increase in retention volume is driven by the laminar flow profile and concomitant diffusion between the layers within the connective tubing of the HPLC system. The process of molecular diffusion alone has been shown to have negligible impact on this effect and is a positive outcome for systems requiring “parking” within sample loops such as in LC x LC systems. However, the deformation of sample plugs due to laminar flow effects were greatest at higher flow rates and in narrower tubing, which could have a significant impact on fast LC technologies such as UHPLC, short and narrow columns, and systems with unavoidable additional tubing lengths. This effect should be considered during method development and transfers between HPLC systems with variable extra column tubing dimensions and especially when utilizing micro columns with non-porous particles or in cases of minimally retentive analytes