An Approach to High Throughput Measurement of Accurate Retention Data in Liquid Chromatography

Efforts to model and simulate various aspects of liquid chromatography (LC) separations (e.g., retention, selectivity, peak capacity, injection breakthrough) depend on experimental retention measurements to use as the basis for the models and simulations. Often these modeling and simulation efforts are limited by datasets that are too small because of the cost (time and money) associated with making the measurements. Other groups have demonstrated improvements in throughput of LC separations by focusing on “overhead” associated with the instrument itself – for example, between-analysis software processing time, and autosampler motions. In this paper we explore the possibility of using columns with small volumes (i.e., 5 mm x 2.1 mm i.d.) compared to conventional columns (e.g., 100 mm x 2.1 mm i.d.) that are typically used for retention measurements. We find that isocratic retention factors calculated for columns with these dimensions are different by about 20%; we attribute this difference – which we interpret as an error in measurements based on data from the 5 mm column – to extra-column volume associated with inlet and outlet frits. Since retention factor is a thermodynamic property of the mobile/stationary phase system under study, it should be independent of the dimensions of the column that is used for the measurement. We propose using ratios of retention factors (i.e., selectivities) to translate retention measurements between columns of different dimensions, so that measurements made using small columns can be used to make predictions for separations that involve conventional columns. We find that this approach reduces the difference in retention factors (5 mm compared to 100 mm columns) from an average of 18% to an average absolute difference of 1.7% (all errors less than 8%). This approach will significantly increase the rate at which high quality retention data can be collected to thousands of measurements per instrument per day, which in turn will likely have a profound impact on the quality of models and simulations that can be developed for many aspects of LC separations.