When scaling an established analytical method across columns packed with different particle sizes and different column configurations (internal diameters and lengths), the amount of time that is required outside of the lab to produce the equivalent method conditions is considerable. When dealing with a gradient method, the calculations required include determining the new flow rate, gradient times, and injection volume. An analyst can perform these calculations manually using the appropriate equations or tools like the Waters Columns Calculator. This app note examines two scaling workflows by first performing a theoretical scale-down experiment manually using the appropriate method scaling equations, and then repeating the experiment using the scaled-down conditions generated by the Waters Columns Calculator. Strong agreement between manual calculations and the results using the Waters Columns Calculator validate the Waters Column Calculator for its use in scaling methods effectively, with a significant improvement in time savings and a reduction in potential calculation errors and/or uncertainties.
Inside the Laboratory: Measuring Molecular Tracers of Emerging Aerosol Sources
October 14th 2024Inside the Laboratory is a joint series with LCGC International and Spectroscopy, profiling analytical scientists and their research groups at universities all over the world. This series spotlights the current chromatographic and spectroscopic research their group is conducting, and the importance of their research in analytical chemistry and specific industries.
Top-down characterization of engineered Bcl-xL proteoforms
October 11th 2024Top-down fragmentation enables rapid characterization of phosphorylated proteins without extensive sample preparation and digestion. In this study, electron capture dissociation (ECD) was used to fragment proteoforms of the cell death-related protein, Bcl-xL. Using these methods, 85–90% sequence coverage was achieved for Bcl-xL proteoforms, allowing for effective localization of phosphorylation within minutes.
Antibody peptide mapping using the new Agilent ExD cell
October 11th 2024Enhanced antibody analysis using electron capture dissociation (ECD) allows for precise glycan localization in low-abundance glycopeptides. This study compares the fragmentation of trastuzumab tryptic digests using ECD and collision-induced dissociation (CID). While CID generates abundant glycan HexNAc ions at 204 m/z, ECD preserves the labile glycan group, enabling accurate site localization.