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UPLC Screening to Preparative HPLC Using CSH Column Technology and Focused Gradients

Reaction screening and purification are essential steps in small molecule pharmaceutical discovery labs.

by Waters


Reaction screening and purification are essential steps in small molecule pharmaceutical discovery labs. Cleaner samples for subsequent synthesis can reduce reaction by-products and facilitate the critical final purification of material for pharmaceutical profiling and toxicology studies.

In this application, we demonstrate a rapid UPLC screening protocol using high and low pH on a charged-surface hybrid (CSH) column and subsequent scale-up to preparative HPLC using focused-gradients. The CSH column is chosen for its improved peak shape for basic compounds, pH range, and retention time stability with routine pH switching. The use of the same stationary phase on the preparative scale maintains the selectivity from the UPLC® screening run, alleviating the need for method re-development. After calculating the focused gradient from the UPLC screening run, the separation is directly transferred to preparative HPLC for rapid purification of the compound of interest. The use of focused gradients in preparative HPLC allows for cleaner purification of the target compound in a shorter run time compared to the full gradient, resulting in higher sample purification throughput.


Samples representative of synthetic reaction products were prepared by acetylating 18 different compounds using pyridine and acetic anhydride.

LC Conditions

Instrument: ACQUITY UPLC® with ToF-MS
Column: ACQUITY UPLC CSH C18 2.1 x 30 mm, 1.7 μm
Part Number: 186005295

Instrument: Autopurification Preparative HPLC system
Column: XSelect™ CSH C18 OBD 19 x 100 mm, 5 μm
Part Number: 186005421


The reactions were first analyzed using a 3-minute UPLC screening protocol at high and low pH, on an ACQUITY UPLC CSH C18 column (Figure 1). Successful reactions were selected for further purification of the target compound using the modifier that was identified as giving the best separation. The retention time of the target compound was then used to calculate the focused gradient for preparative HPLC.

Copyright; Xselect, UPLC, ACQUITY UPLC, The Science of what’s possible

Figure 1. A UPLC screening result at high and low pH. The high pH separation is selected for purification of the target peak at 1.06 min.

By altering the original screening gradient to a shallower ‘focused’ gradient around the peak of interest, impurities can be selectively pulled away to generate a cleaner purified sample (Figure 2). The use of focused gradients minimizes preparative HPLC runtimes as time is spent on the separating the peak of interest rather than the entire sample.

Figure 2. The target peak at 1.06 min was transferred from UPLC to prep HPLC using a focused-gradient. Mass-directed purification of the target peak results in a pure product, confirmed using UPLC.


A 3-minute UPLC-MS method that reliably screens at high and low pH was developed using an ACQUITY UPLC CSH C18 column. The separation was transferred to preparative HPLC using the same stationary phase, alleviating the need to re-develop the method at the preparative scale. The use of focused gradients shortens runtimes and increases sample throughput by selectively focusing the separation around the peak of interest.