uR2 2.1x100mm UHPLC Column
The LudgerSep uR2 2.1x100mm UHPLC column has been developed for the analysis of sialic acids labeled with DMB (1,2-diamino-4,5 methylenedioxybenzene.2HCl) using the LudgerTag DMB Sialic Acid Labeling Kit.
The LudgerSep uR2 column is designed for use with the latest generation of UHPLC instruments capable of withstanding high flow pressures and fast sample analyses. In order to take advantage of the high resolving power of sub 3 μm particle size containing columns, we recommend keeping sample injection volumes at or below 5 μL and minimising system void volumes. Ideally use full loop injection. Tubing should be narrow bore (about 0.13 mm diameter or less) and detector flow cell volumes should be 10 μl or less. Although an example chromatogram is shown in the product guide, retention times will vary dependent on the UHPLC system used.
Overview of the Procedure
Release of Sialic Acids
The release of sialic acids from the glycoprotein occurs by a mild acid hydrolysis. Samples and controls are incubated with 2M acetic acid at 80°C for 2 hours.
Sialic Acid Labeling Reaction
The released silica acids are fluorescently labelled with 1,2-diamino-4,5-methylenedioxybenzene-2HCl (DMB). The acid hydrolysed samples and process controls plus Ludger sialic acid standards: Neu5Ac, Neu5Gc, sialic acid reference panel, and Neu5,9Ac2 if using, are incubated with DMB at 50°C for 3 hours.
Analysis via HPLC or UHPLC
A standard curve is calculated using the Neu5Ac and Neu5Gc standards. The sialic acid reference panel and Neu5,9Ac2 are used for sialic acid identification. Analysis can be performed using the LudgerSep R1x150mm column for HPLC or via UHPLC using a LudgerSep uR2x100mm column.
DMB Labelled Sialic Acid Reference Panel Run on the LudgerSep uR2 2.1x100mm UHPLC column.
Peaks: 1 = Neu5Gc; 2 = Neu5Ac; 3 = Neu5,7Ac2; 4 = Neu5Gc,9Ac; 5 = Neu5,8Ac2; 6 = Neu5,9Ac2; 7 = Neu5,x,xAc3 (where x is an unknown acetyl position); * = Reagent
uR2 2.1x100mm UHPLC Column Specifications
Particles: 1.9 µm silica derivatized with octadecylsiliane coating. 175 Angstrom pore size.
Column dimensions: 2.1mm x 100mm
Column volume: 0.34 mls
Typical flow rate: 0.1 – 0.6 ml/min
Pressure limit: 15,000 psi
Column tube: Stainless steel, Valco compatible end fittings
pH compatibility: pH 1 to pH 11