Tunable temperature responsive liquid chromatography through thiolactone-based immobilization of poly(N-isopropylacrylamide).

A straightforward and efficient functionalization of aminopropylsilica with polymeric structures is described for the development of temperature responsive stationary phases applicable in purely aqueous liquid chromatography. The immobilization of the thermoresponsive polymers involves a thiolactone-based ring opening using the primary amines in aminopropylsilica, with a simultaneous one-pot, thiol-ene functionalization with an acrylate of choice. This mild, straightforward and modular grafting process results in high polymer coupling yields. By variation of the acrylate for the thiol-ene reaction, different stationary phases can be readily obtained. Two stationary phases as a result of the modular modification of aminopropylsilica were evaluated with test mixtures of hydrophobic analytes and a mixture of di- and tripeptides. Analyses using the 5µm material packed in 10cm×4.6mm columns revealed high hydrophobic retention, which proved adaptable as a function of the temperature in aqueous mobile phases. High versus low retention were obtained at temperatures above and below the lower critical solution temperature of the polymer, respectively. Moreover, the columns depict potential for diastereoisomeric peptide separation. Finally, the lower retention, observed when using PEGylated silica, illustrates the potential of the approach for modular stationary phase tuning.

Comprehensive two-dimensional liquid chromatography coupled to the ABTS radical scavenging assay: a powerful method for the analysis of phenolic antioxidants.

The on-line combination of comprehensive two-dimensional liquid chromatography (LC × LC) with the 2,2'-azino-bis(3-ethylbenzothiazoline)-6 sulphonic acid (ABTS) radical scavenging assay was investigated as a powerful method to determine the free radical scavenging activities of individual phenolics in natural products. The combination of hydrophilic interaction chromatography (HILIC) separation according to polarity and reversed-phase liquid chromatography (RP-LC) separation according to hydrophobicity is shown to provide much higher resolving power than one-dimensional separations, which, combined with on-line ABTS detection, allows the detailed characterisation of antioxidants in complex samples. Careful optimisation of the ABTS reaction conditions was required to maintain the chromatographic separation in the antioxidant detection process. Both on-line and off-line HILIC × RP-LC-ABTS methods were developed, with the former offering higher throughput and the latter higher resolution. Even for the fast analyses used in the second dimension of on-line HILIC × RP-LC, good performance for the ABTS assay was obtained. The combination of LC × LC separation with an on-line radical scavenging assay increases the likelihood of identifying individual radical scavenging species compared to conventional LC-ABTS assays. The applicability of the approach was demonstrated for cocoa, red grape seed and green tea phenolics.

Deconvolution of overlapping spectral polymer signals in size exclusion separation-diode array detection separations by implementing a multivariate curve resolution method optimized by alternating least square.

Peaks eluting from a size exclusion separation (SEC) are often not completely baseline-separated due to the inherent dispersity of the polymer. Lowering the flow rate is sometimes a solution to obtain a better physical separation, but results in a longer retention time, which is often not desirable. The chemometrical deconvolution method discussed in this work provides the possibility of calculating the contribution of each peak separately in the total chromatogram of overlapping peaks. An in-house-developed MATLAB script differentiates between compounds based on their difference in UV-spectrum and retention time, using the entire 3D retention time UV-spectrum. Consequently, the output of the script offers the calculated chromatograms of the separate compounds as well as their respective UV-spectrum, of which the latter can be used for peak identification. This approach is of interest to quantitate contributions of different polymer types with overlapping UV-spectra and retention times, as is often the case in, for example, copolymer or polymer blend analysis. The applicability has been proven on mixtures of different polymer types: polystyrene, poly(methyl methacrylate) and poly(ethoxyethyl acrylate). This paper demonstrates that both qualitative and quantitative analyses are possible after deconvolution and that alternating concentrations of adjacent peaks do not significantly influence the obtained accuracy.