Characterization of a High Throughput Approach for Large Scale Retention Measurement in Liquid Chromatography

Abstract

Many contemporary challenges in liquid chromatography - such as the need for “smarter” method development tools, and deeper understanding of chromatographic phenomena - could be addressed more efficiently and effectively with larger volumes of experimental retention data than we have been accustomed to historically. The paucity of publicly accessible, high-quality measurements has been due, at least in large part, to the high cost in time and resources associated with traditional retention measurement approaches. Recently we described an approach to improve the throughput of such measurements by using very short columns (typically 5 mm), while maintaining measurement accuracy. In this paper we present a perspective on the characteristics of results obtained using this approach using a dataset containing about 13,000 retention measurements, and describe an approach to sample introduction that improves upon the prior work. The dataset is comprised of results for 35 different small molecules, nine different stationary phases, and several mobile phase compositions for each analyte/phase combination. During the acquisition of these data, we have interspersed repeated measurements of a small number of compounds for quality control purposes. The data from these measurements not only enable detection of “out of control” measurements, but also assessment of the repeatability and reproducibility of retention measurements over time. For retention factors greater than 1, the mean relative standard deviation (RSD) of replicate (typically n=5) measurements is 0.4%, and the standard deviation of RSDs is 0.4%. Most differences between selectivity values measured six months apart for 15 non-ionogenic compounds were in the range of +/- 1%, indicating good reproducibility. A critically important observation from these analyses is that selectivity (defined here as retention of a given analyte relative to the retention of a reference compound; kx/kref) is a much more consistent measure of retention over time (months) compared to the retention factor alone. While this work and dataset also highlights the importance of stationary phase stability over time for achieving reliable retention measurements, we are nevertheless optimistic that this approach will enable the compilation of large databases (>> 10,000 measurements) of retention values over long time periods (years), which can in turn be leveraged to address some of the most important contemporary challenges in liquid chromatography. All of the data discussed in the manuscript are provided as Supplemental Information.

Improved hydrophobic subtraction model of reversed-phase liquid chromatography selectivity based on a large dataset with a focus on isomer selectivity

“…5) for specific solute/stationary phase pairs. Much more work is needed to understand the drivers of this uncertainty (e.g., stationary phase drift over time [26]) so that we can work to minimize it in the future.…”

“…As described in ref. [26], samples were introduced to the mobile phase stream using a "feed injection" approach, and the injection volume was 150 nL. The solutes and stationary phases used are listed in the supplementary materials in Tables S1 and S2.…”

“…The dimensions of all these columns are given in Table S2. The details associated with the measurement steps and implementation of correction factors were described previously [25,26].…”

“…The full retention dataset used in this work (43,329 measurements) is provided as Supplemental Information in the file "WC_second_kernel_database.xlsx", along several files containing quality control (QC) data as outlined in the Supplemental Information. Note that a subset of the full dataset shared here was published previously (12,319 measurements) [26], and we provide them again here simply for the convenience of the reader.…”

“…Therefore, in the present study, we have attempted to address the primary limitations of the previous studies: 1) the HSM1 dataset is composed of retention measurements made with just one mobile phase composition (50/50 ACN/buffer), which precludes any direct application of the model to gradient elution conditions; 2) the buffer used for HSM1 contains phosphates, which are incompatible with mass spectrometric detection -an essential tool in the analysis of pharmaceuticals; and 3) the probe solutes have been limited to a small number of relatively simple compounds. In this work, we have produced a large set of retention measurements using our highthroughput method for characterizing retention described previously [25][26][27]. The new dataset includes 86 solutes and 13 stationary phases, and retention has been measured at multiple mobile phase compositions for each compound/column combination, for a total of about 40,000 measurements.…”