Physicochemical modelling of the retention mechanism of temperature-responsive polymeric columns for HPLC through machine learning algorithms.

Temperature-responsive liquid chromatography (TRLC) offers a promising alternative to reversed-phase liquid chromatography (RPLC) for environmentally friendly analytical techniques by utilizing pure water as a mobile phase, eliminating the need for harmful organic solvents. TRLC columns, packed with temperature-responsive polymers coupled to silica particles, exhibit a unique retention mechanism influenced by temperature-induced polymer hydration. An investigation of the physicochemical parameters driving separation at high and low temperatures is crucial for better column manufacturing and selectivity control. Assessment of predictability using a dataset of 139 molecules analyzed at different temperatures elucidated the molecular descriptors (MDs) relevant to retention mechanisms. Linear regression, support vector regression (SVR), and tree-based ensemble models were evaluated, with no standout performer. The precision, accuracy, and robustness of models were validated through metrics, such as r and mean absolute error (MAE), and statistical analysis. At 45 ∘ C , logP predominantly influenced retention, akin to reversed-phase columns, while at 5 ∘ C , complex interactions with lipophilic and negative MDs, along with specific functional groups, dictated retention. These findings provide deeper insights into TRLC mechanisms, facilitating method development and maximizing column potential.

Temperature-responsive comprehensive two-dimensional liquid chromatography coupled to high resolution mass spectrometry for the elucidation of the oxidative degradation processes of chemicals of environmental concern.

This study explores the possibilities offered by temperature-responsive liquid chromatography (TRLC) based comprehensive 2-dimensional liquid chromatography in combination with reversed-phase liquid chromatography (RPLC) for the analysis of degradation products formed upon oxidative treatment of persistent organic pollutants, in this case exemplified through carbamazepine (CBZ). The TRLC×RPLC combination offers the possibility to overcome peak overlap and incomplete separation encountered in 1D approaches, while the transfer of the purely aqueous mobile phase leads to refocusing of all analytes on the second dimension column. Consequently, this allows for about method-development free and hence, easier LC×LC. The study focuses on the oxidative degradation of CBZ, a compound of environmental concern due to its persistence in water bodies. The TRLC×RPLC combination effectively separates and identifies CBZ and its degradation products, while offering improved selectivity over the individual TRLC or RPLC separations. This allows gathering more understanding of the degradation cascade and allows real-time monitoring of the appearance and disappearance of various degradation products. The compatibility with high-resolution mass spectrometry is last shown, enabling identification of 21 CBZ-related products, nine of which were not previously reported in CBZ degradation studies. The approach's simplicity, optimization-free aspects, and ease of use make it a promising tool for the analysis of degradation pathways in environmental contaminants.

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 efficiencies 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.

Hyphenating temperature gradient elution with refractive index detection through temperature-responsive liquid chromatography.

Refractive index detection (RID) is attractive because it allows approaching the benefits of universal detection with liquid chromatography, by which ideally standard independent calibration and hence compound independent quantification becomes possible. Nevertheless, the implementation of RID has remained limited as it offers poor detection sensitivity while only being compatible with isocratic mobile phases. The implementation of compositional solvent gradients has remained prohibitively challenging in commercial HPLC-RID systems due to the resulting drastic alterations in refractive index and extreme baseline drift. While the refractive index is also highly dependent on temperature, more leeway appears possible to mitigate the problem, particularly when the used temperature gradients can be limited. Temperature-responsive liquid chromatography (TRLC) allows obtaining isocratic reversed phase type of separations, whereby retention is modulated via temperature changes âˆ¼ 15 °C-20 °C above and below the polymer conversion temperature. Elution profiles, reminiscent of what can be obtained with solvent gradients in conventional RPLC, can then be obtained by enacting downwards temperature gradients on the columns. This work comprises a proof-of-principle to illustrate the possibilities of combining thermal gradient TRLC with RID. The observed baseline drift appeared thereby very minor (<5 nRIU min-1), and hence easily controllable. Short chain fatty acids are used as representative compounds to assess this new approach. Overlapping calibration lines are accordingly obtained for all fatty acids between butyric and decanoic acid.

Comprehensive two-dimensional temperature-responsive × reversed phase liquid chromatography for the analysis of wine phenolics.

Phenolic compounds are an interesting class of natural products because of their proposed contribution to health benefits of foods and beverages and as a bio-source of organic (aromatic) building blocks. Phenolic extracts from natural products are often highly complex and contain compounds covering a broad range in molecular properties. While many 1D-LC and mass spectrometric approaches have been proposed for the analysis of phenolics, this complexity inevitably leads to challenging identification and purification. New insights into the composition of phenolic extracts can be obtained through online comprehensive two-dimensional liquid chromatography (LC × LC) coupled to photodiode array and mass spectrometric detection. However, several practical hurdles must be overcome to achieve high peak capacities and to obtain robust methods with this technique. In many LC × LC configurations, refocusing of analytes at the head of the 2D column is hindered by the high eluotropic strength of the solvent transferred from the 1D to the 2D, leading to peak breakthrough or broadening. LC × LC combinations whereby a purely aqueous mobile phase is used in the 1D and RPLC is used in the 2D are unaffected by these phenomena, leading to more robust methods. In this contribution, the combination of temperature-responsive liquid chromatography (TRLC) with RPLC is used for the first time for the analysis of phenolic extracts of natural origin to illustrate the potential of this alternative combination for natural product analyses. The possibilities of the combination are investigated through analysis of wine extracts by TRLC × RPLC-DAD and TRLC × RPLC-ESI-MS.