Orthogonal Selectivity of ZirChrom®-CARB
Method development in reversed-phase liquid chromatography (RPLC) can be challenging for structurally similar compounds.
Dr. Bingwen Yan and Dr. Clayton McNeff, ZirChrom Separations, Inc.
Method development in reversed-phase liquid chromatography (RPLC) can be challenging for structurally similar compounds. Validation methods often require a column of orthogonal selectivity be used to ensure no impurities are missed, carbon-clad zirconia phase offers dramatically different chromatographic selectivity when compared to traditional silica-based bonded phases. This note shows selectivity comparison of the zirconia based, carbon-clad ZirChrom® -CARB HPLC column versus a typical silica bonded phase for 22 nonelectrolyte solutes with varying chemical properties.
Method development in RPLC has traditionally been difficult for molecules which are geometric isomers or structurally very similar. In bonded-phase silicas, the partition mechanism responsible for retention in RPLC often does not offer adequate chemical selectivity for such compounds. On the other hand, carbon-based phases provide retention in RPLC through an adsorption mechanism which often increases the chromatographic selectivity for these types of compounds and dramatically increases the chances of resolving pairs of these analytes.
In addition to this enhanced and often orthogonal chromatographic selectivity, carbon-based phases also offer increased chemical and thermal stability of the stationary phase. Zichrom Separations, Inc. has developed ZirChrom®-CARB using zirconia as a stationary phase support, and patented chemical vapor deposition technology to produce a carbon-clad stationary phase suitable for use in reversed-phase liquid chromatography. The graphitized carbon surface is extremely resistant to chemical and thermal attack. The authors have run mobile phases at very high pH (1 M NaOH), very low pH (0.5 M HNO3), and at elevated temperature (200 °C) and have not observed loss of stationary phase. This unique chemistry results in a very selective phase with no detectable bleed by LC–MS.
Experimental
22 non-electrolyte solutes were injected onto two HPLC columns of equivalent configuration; 1) ZirChrom®-CARB, 3 µm particle size and 2) a leading traditional silica based bonded phase (ODS), 3 µm particle size. For both HPLC columns the k' for each solute was calculated from the resulting data. Each k' was then converted to log k' and graphed, see Figure 1.
Figure 1
Column: ZirChrom®-CARB, 50 mm x 4.6 mm i.d.
(Part Number: ZR01-0546)
Mobile Phase: 40/60 acetonitrile/water
Temperature: 30 °C with Metalox™ 200-C column heater
Flow Rate: 1 mL/min
Injection Vol.: 5 µL
Pressure Drop: 50 bar
Detection: UV at 254 nm
This method can be tailored to your specific application needs. ZirChrom technical support can help to optimize and transfer this method to your site. Please contact ZirChrom technical support at 1-866-STABLE-1 or support@zirchrom.com for details.
ZirChrom phases offer unique selectivity, high efficiency, and excellent chemical and thermal stability.
Visit www.zirchrom.com for more application notes using ultrastable, high efficiency ZirChrom columns.
ZirChrom Separations, Inc.
617 Pierce Street, Anoka, MN 55303
tel. 1-866-STABLE-1
Email: support@zirchrom.com; Website: www.zirchrom.com
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.
Identification of Amino Acid Isomers Using Electron Capture Dissociation
October 11th 2024Electron capture dissociation produces distinct fragments of amino acid side chains, enabling the identification of isomeric amino acids such as leucine and isoleucine. This application note demonstrates the isomer identification workflow for peptides and intact proteins using the new Agilent ExD cell and ExDViewer software for fragment analysis.
Trends, Best Practices, and Analytical Challenges in Chemical Characterization of Medical Devices
October 7th 2024Chemical characterization of medical devices, including drug-device combination products, is crucial for ensuring regulatory compliance and patient safety by identifying and quantifying chemicals that may interact with the human body. This paper explores current trends, best practices, and regulatory developments in extractables and leachables (E&L) testing for medical devices.
