On-column modification for the creation of temperature-responsive stationary phases

Temperature-responsive liquid chromatography (TRLC) offers an alternative for retention and selectivity optimisation in HPLC. This approach thereby exploits temperature gradients on stimuli-responsive stationary phases and forfeits the necessity for solvent gradients, allowing analyses to be performed using aqueous mobile phases. Consequently, it can be employed as a green alternative to reversed-phase separations. However, current production to obtain temperature-responsive columns inherently require dedicated column packing processes with polymer-modified particles. To facilitate the development of temperature-responsive phases, a flow-through modification procedure was developed allowing on-column modification of aminopropyl silica columns. Three columns were manufactured using this novel flow-through approach, which achieved identical column effi-ciencies compared to existing TRLC column. Demonstrating the possibility of bypassing the dedicated packing processes without losing efficiency. Additionally, it was observed that flow-through produced columns yielded higher retention at elevated temperatures despite their reduced carbon load. Further investigation of the carbon load revealed the presence of stationary phase gradients, whose influence was studied via novel developed retention experiments, which revealed a negligible change reduction in retention upon a change of polymer modification from 19.8% to 14.5%. However, further decrease from 14.5% to 12.3% resulted in a larger change. Interestingly, a further enhancement in apparent plate numbers was observed when operating the column under a reversed flow, yielding an increasing stationary phase gradient. This on-column modification procedure demonstrates the potential for modification of existing (commercial) packed columns to achieve temperature-responsive phases without loss of efficiency or retention. Thus, not only facilitating accessibility to temperature-responsive phases, but also aiding with development of further generations of temperature-responsive phases by removing the need for packing optimisation. Additionally, a novel experiment was set up to easily investigate the effect of inhomogeneous stationary phases retention.