Advances in ultra-high-pressure and multi-dimensional liquid chromatography instrumentation and workflows

The present contribution discusses recent advances in ultra-high-pressure liquid chromatography (UHPLC) and multi-dimensional liquid chromatography (MDLC) technology. First, new developments in UHPLC column technology and system design are highlighted. The latter includes a description of a novel injector concept enabling method speed-up, emerging detectors, and instrument diagnostics approaches. Next, online MDLC workflows are reviewed and advances in modulation technology are highlighted. Finally, key applications published in 2020 are reviewed

The Determination of a Threshold Level of Exogenous Methyl Jasmonate Effecting an Increase in Peroxidase Activity in Protoplasts of Avena Sativa

Methyl jasmonate plays a dual role in plant systems by participating in both developmental and defense mechanisms. Recent studies have shown that methyl jasmonate can specifically alter gene expression, while wounding and specific elicitors can cause a buildup of methyl jasmonate itself. Peroxidase enzymes are ubiquitous to all plant systems and have long been associated with plant stress and defense responses. The objective of this research was to establish a threshold concentration of methyl jasmonate effecting a maximum increase in peroxidase activity less than twenty-four hours after treatment. While previous experiments have explored the activity of methyl jasmonate on peroxidase activity using whole leaf tissue, we developed a system for testing peroxidase activity in oat protoplasts after isolation and subsequent treatment and incubation in a suitable culture media

Analytically-speaking-podcast podcast "sometimes negative results turn out to be the most interesting ones": Adrian Clarke interviewed by Dwight Stoll.

Analytically Speaking podcast from LCGC, episode 6 ""sometimes negative results turn out to be the most interesting ones" , with Adrian Clarke interviewed by Dwight Stoll. addresses important issues in separation science. Topics include, how strated in science, my current role, responsibilities and scientific interests. new analytical techniques, methods, and approaches; the latest trends; advances in instrument and software technology; recent papers in the scientific literature and their applicability; challenges and solutions for pharmaceutical analysis

But my peaks are not Gaussian! Part 3: Physicochemical causes of peak tailing

Although symmetric peaks with Gaussian shapes are predicted by models of the chromatographic process, "perfect peaks" are not observed often outside of textbooks. Several physico-chemical phenomena can lead to asymmetric peak shapes, including analyte adsorption to different types of sites within the stationary phase, and overload tailing, which may involve a variety of factors. Understanding these phenomena can help identify whether the cause of asymmetry is most likely to have a physical or chemical origin, which in turn dictates which troubleshooting steps to start with when dealing with poor peak shapes

Smart Templates for peak pattern matching with comprehensive two-dimensional liquid chromatography

Comprehensive two-dimensional liquid chromatography (LC × LC) generates information-rich but complex peak patterns that require automated processing for rapid chemical identification and classification. This paper describes a powerful approach and specific methods for peak pattern matching to identify and classify constituent peaks in data from LC × LC and other multidimensional chemical separations. The approach records a prototypical pattern of peaks with retention times and associated metadata, such as chemical identities and classes, in a template. Then, the template pattern is matched to the detected peaks in subsequent data and the metadata are copied from the template to identify and classify the matched peaks. Smart Templates employ rule-based constraints (e.g., multispectral matching) to increase matching accuracy. Experimental results demonstrate Smart Templates, with the combination of retention-time pattern matching and multispectral constraints, are accurate and robust with respect to changes in peak patterns associated with variable chromatographic conditions

The Effects of Wounding and Ethylene on Cellular Fatty Acid Composition of Avena Sativa

Plant defense responses to wounding, pathogen attack, and environmental stresses often involve the production of various intermediates of the octadecanoid pathway, which are derived from the fatty acid linolenic acid. Ethylene is a plant hormone that has also been implicated in defense responses. The goal of this project has been to determine if wounding causes changes in the fatty acid composition of plants and to determine whether ethylene plays a role in this response. Controlled and wounded nine-day old Avena sativa (oats) plants were treated with ethylene, norbomadiene or silver thiosulfate. Norbomadiene and silver thiosulfate inhibit the action of endogenous ethylene. The oat leaves were harvested at 0, 1,4, 8, and 22 hours following wounding and treatment. Lipids were then extracted from leaves and the lipids were fractionated using solid phase extraction. The methyl esters of the fatty acids in these fractions were prepared by transesterification with sulfuric acid in methanol. The resulting fatty acid methyl esters will be analyzed using gas chromatography with methyl heptadecanoate as an internal standard

Tips, Tricks, and Troubleshooting for Separations of Biomolecules, Part I: Contemporary Reversed-Phase Protein Separations

Several new materials and columns have been introduced in recent years for reversed-phase separations of proteins. How do I know which one to choose, and which separation conditions will be best for my protein separation

Tips, Tricks, and Troubleshooting for Separations of Biomolecules, Part 1: Contemporary Reversed-Phase Protein Separations

Several new materials and columns have been introduced in recent years for reversed-phase separations of proteins. How do I know which one to choose, and which separation conditions will be best for my protein separation

Experimental design and re-parameterization of the Neue-Kuss model for accurate and precise prediction of isocratic retention factors from gradient measurements in reversed phase liquid chromatography

The present work describes a re-parameterization of the Neue Kuss (NK) model for describing retention in liquid chromatography, and this re-parameterized model is used fit a large set of isocratic retention measurements with improved convergence properties relative to the original parameterization of the model. Next, an experimental design for retention measurements made using mobile phase gradient elution conditions is proposed for the purpose of obtaining accurate and precise NK parameters. Simulated retention data for mobile phase gradient elution conditions with two different levels of noise, as well as an essentially zero noise level were fit with the re-parameterized model. The results showed that the re-parameterized fits yielded average (absolute value) prediction errors for the parameters at the highest noise level of 7.16 % for S1,ref, 17.5 % for S2,ref and 6.19 % for kref. These errors were significantly smaller than those for the original parameterization of the NK model, where the errors were 23.2 % for S1, 24.9 % for S2 and 159 % for kw. Furthermore, isocratic retention factors predicted using these model parameters were found to have an average magnitude of error of 0.509 % for the re-parameterized model, as opposed to 6750 % for the model with the original parameterization. A further test of this approach was carried out for independent experimental measurements for five solutes and a C18 column. The average magnitude of error of the isocratic retention factors predicted from parameters obtained from fits of gradient data was 1.59 %, provided that the range of organic solvent compositions that the solute sampled in the mobile phase gradient experiments was consistent with the isocratic experiments. These results indicate that the reparameterization of the NK model allows for significant improvements in the fitting process, and that the proposed experimental design allows for NK parameters to be extracted from mobile phase gradients experiment that predict isocratic retention factors with an accuracy on the order of 1%